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Chem 333 – Metabolism Lecture Notes Michael Palmer Department of Chemistry University of Waterloo Waterloo, Ontario, Canada
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Chem 333 – Metabolism
Lecture Notes
Michael PalmerDepartment of ChemistryUniversity of Waterloo
Waterloo, Ontario, Canada
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Preface
These notes are for use with my 3rd year undergraduate class on metabolism.The focus is on human metabolism, and more specifically on human energy me-tabolism. Apart from the pathways and reactions, several aspects of hormonalregulation and some medical correlations are also included. The entire texthas been prepared by myself. The same applies to most figures; exceptions areindicated. With respect to my own material, you are welcome to use it in yourown not-for-profit teaching materials as you see fit, provided that proper creditis given.
These notes are currently in their 4th edition. In this edition, some errorshave been corrected, yet likely some more remain to be discovered, and newones may have been introduced. I therefore welcome corrections and sug-gestions for improvement, no matter whether you’re a colleague, a student,or simply an interested reader. Please send email to [email protected] you.
Contents
Chapter 1 Introduction 1
1.1 Motivation: Why would you study metabolism? . . . . . . . . . . 11.2 Catabolic and anabolic reactions . . . . . . . . . . . . . . . . . . . 11.3 Metabolic diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.4 Types of foodstuffs . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.5 The digestive tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.6 What’s next? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Chapter 2 Refresher 16
2.1 How enzymes work: Active sites and catalytic mechanisms . . 162.2 Classification of Enzymes and enzyme reactions . . . . . . . . . 182.3 Energetics of enzyme-catalyzed reactions . . . . . . . . . . . . . 192.4 The role of ATP in enzyme-catalyzed reactions . . . . . . . . . . 212.5 Regulation of enzyme activity . . . . . . . . . . . . . . . . . . . . 23
Chapter 3 Glycolysis 26
3.1 Overview of glucose metabolism . . . . . . . . . . . . . . . . . . . 263.2 The place of glycolysis in glucose degradation . . . . . . . . . . 273.3 Reactions in glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . 283.4 Mechanisms of enzyme catalysis in glycolysis . . . . . . . . . . 283.5 Energy-rich functional groups in substrates of glycolysis . . . . 353.6 Function of glycolysis under anaerobic conditions . . . . . . . . 363.7 Transport and utilization of glucose . . . . . . . . . . . . . . . . 37
Chapter 4 Catabolism of sugars other than glucose 40
4.1 Metabolism of sucrose and fructose . . . . . . . . . . . . . . . . . 414.2 Metabolism of lactose and galactose . . . . . . . . . . . . . . . . 424.3 The polyol pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
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iv CONTENTS
Chapter 5 Pyruvate dehydrogenase and the citric acid cycle 48
5.1 Pyruvate dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . 48
5.2 PDH: Catalytic mechanisms . . . . . . . . . . . . . . . . . . . . . . 52
5.3 Regulation of pyruvate dehydrogenase . . . . . . . . . . . . . . . 52
5.4 The citric acid cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
5.5 Reactions in the citric acid cycle . . . . . . . . . . . . . . . . . . . 56
5.6 Regulation of the citric acid cycle . . . . . . . . . . . . . . . . . . 59
Chapter 6 The respiratory chain 60
6.1 ATP synthesis can be separated from electron transport . . . . 62
6.2 The electron transport chain . . . . . . . . . . . . . . . . . . . . . 65
6.3 ATP synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
6.4 The ATP yield of oxidative glucose degradation . . . . . . . . . 79
6.5 Shuttle systems for the NADH re-oxidation . . . . . . . . . . . . 80
6.6 Regulation of the respiratory chain . . . . . . . . . . . . . . . . . 82
Chapter 7 Gluconeogenesis 85
7.1 Reactions in gluconeogenesis . . . . . . . . . . . . . . . . . . . . . 86
7.2 Glucogenic amino acids and gluconeogenesis . . . . . . . . . . . 88
7.3 Regulation of gluconeogenesis . . . . . . . . . . . . . . . . . . . . 88
7.4 Energy balance of gluconeogenesis . . . . . . . . . . . . . . . . . 92
7.5 The Cori cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
7.6 The glyoxylate cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
7.7 Additional roles of gluconeogenetic enzymes . . . . . . . . . . . 94
Chapter 8 Glycogen metabolism 97
8.1 Glycogen synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
8.2 Glycogen degradation . . . . . . . . . . . . . . . . . . . . . . . . . . 101
8.3 Glycogen storage diseases . . . . . . . . . . . . . . . . . . . . . . . 101
8.4 Regulation of glycogen metabolism . . . . . . . . . . . . . . . . . 103
Chapter 9 The hexose monophosphate shunt 105
9.1 Reactions in the hexose monophosphate shunt . . . . . . . . . . 106
9.2 Mechanisms of transketolase and transaldolase . . . . . . . . . 110
9.3 Why do we need both NADH and NADPH? . . . . . . . . . . . . . 111
9.4 Alternative sources of NADPH . . . . . . . . . . . . . . . . . . . . 111
9.5 Uses of NADPH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
9.6 Glucose-6-phosphate dehydrogenase deficiency . . . . . . . . . 115
CONTENTS v
Chapter 10 Triacylglycerol metabolism 11810.1 Utilization of dietary triacylglycerol . . . . . . . . . . . . . . . . . 12010.2 Utilization of fatty acids: β-Oxidation . . . . . . . . . . . . . . . . 12410.3 Triacylglycerol utilization . . . . . . . . . . . . . . . . . . . . . . . 12810.4 Ketone bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13010.5 Fatty acid synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 13310.6 Pharmacological inhibition of fatty acid synthase . . . . . . . . 139
Chapter 11 Cholesterol metabolism 14111.1 Uptake and transport of cholesterol . . . . . . . . . . . . . . . . . 14211.2 Distribution of cholesterol from the liver to the periphery . . . 14211.3 Transport of excess cholesterol to the liver . . . . . . . . . . . . 14311.4 Esterification of cholesterol . . . . . . . . . . . . . . . . . . . . . . 14411.5 Synthesis of cholesterol . . . . . . . . . . . . . . . . . . . . . . . . 14411.6 The endoplasmic reticulum in steroid synthesis . . . . . . . . . 14611.7 7-Dehydrocholesterol and vitamin D3 . . . . . . . . . . . . . . . . 14811.8 Regulation of cholesterol synthesis . . . . . . . . . . . . . . . . . 14911.9 Cholesterol in atherosclerosis . . . . . . . . . . . . . . . . . . . . 15111.10 Therapy of hypercholesterolemia . . . . . . . . . . . . . . . . . . 152
Chapter 12 Amino acid metabolism 15412.1 Overview of amino acid degradation . . . . . . . . . . . . . . . . 15512.2 Transamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15612.3 The urea cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15812.4 Auxiliary reactions in nitrogen transport and elimination . . . 16112.5 Degradative pathways of individual amino acids . . . . . . . . . 16512.6 Hereditary enzyme defects in amino acid metabolism . . . . . 170
Chapter 13 Hormonal regulation of metabolism 17613.1 Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17613.2 Glucagon and epinephrine . . . . . . . . . . . . . . . . . . . . . . . 18413.3 Glucocorticoids and thyroid hormones . . . . . . . . . . . . . . . 18613.4 Leptin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Chapter 14 Diabetes mellitus 18914.1 The role of insulin and the causation of diabetes . . . . . . . . . 19014.2 Effects of insulin deficiency on carbohydrate metabolism . . . 19014.3 Insulin deficiency and lipid metabolism . . . . . . . . . . . . . . 19214.4 Laboratory findings and clinical symptoms in acute type I
diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19214.5 The cause of β-cell destruction in type I diabetes . . . . . . . . 19414.6 Why do diabetic patients lose glucose in the urine? . . . . . . . 19414.7 Treatment of type I diabetes . . . . . . . . . . . . . . . . . . . . . 19614.8 Long-term complications of diabetes mellitus . . . . . . . . . . . 197
vi CONTENTS
14.9 Glucose assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19914.10 Diabetes type II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20114.11 Diabetic coma and comatose diabetics . . . . . . . . . . . . . . . 20114.12 Other forms of diabetes . . . . . . . . . . . . . . . . . . . . . . . . 20214.13 The End . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Chapter 1
Introduction
1.1 Motivation: Why would you study metabolism?
Long answer: You have a generous and warm character, and you have spenttoo much time in front of the family TV set. You are therefore determinedto become a famous doctor and save many, many lives every hour of the day,without asking anything in compensation but the admiring gaze of the populace,and may be a Rolls Royce. Metabolism is an ever so tiny part of the vastknowledge you have set out to master in order to fulfill your destiny.
Wrong? Try this one: You have the inquisitive mind of a Sherlock Holmesand the financial savvy of a Howard Hughes, and you have determined thatsoaking medical doctors for damages is the best road to wealth and fame.Understanding the biochemical basis of medicine will help you to stun youraudiences in court and grind the defendants and their counsels into the dust.
Wrong again? Then try the short answer: You want to pass your exam.
1.2 Catabolic and anabolic reactions
Metabolism is a central theme in biochemistry. Metabolism keeps cells andorganisms alive, by giving them the energy to carry on and the building blocksrequired for growth and propagation. The metabolism of animals and humans,which depend entirely on foodstuffs, can be divided into catabolic and anabolicreactions.
The term catabolic means the same as degradative, but it is Greek andtherefore sounds a whole lot more erudite and scholarly. Key functions ofcatabolic reactions are
1
2 1 Introduction
Foodstuffs small intermediates
complexbiomolecules
CO2 + H2Osmall intermediates
ADP+Pi
NADP+
ATPNADPH+H+
O2
Figure 1.1 A broad outline of human metabolism. Foodstuffs, which are mostly ma-cromolecules, are broken down in catabolic reactions to small intermediates, which inturn are in part further degraded to yield energy (ATP) and reducing power (NADPH).Some of the intermediates thus produced are used in anabolic reactions for the synthe-sis of new macromolecules. These may in turn be recycled and used as substrates forthe release of small intermediates.
1. accumulation of energy in the form of ATP,2. regeneration of reducing power (NADPH), and3. production of building blocks for anabolic metabolism.A large share of the substrates broken down in catabolism are being used
for producing ATP, the “electric energy” of the cell. Just as electricity canbe used to drive just about any household job, ATP is used for nearly everyenergy-requiring task in cell biology. This includes
1. cell motility, particularly in muscle cells,2. active transport across membranes, e.g. Na+/K+-ATPase and other ion
pumps. Ion pumps are largely responsible for the conspicuous energyrequirements of brain and kidneys,
3. anabolic metabolism.Because of its key role in the life of the cell, we will devote a good deal of timeto the metabolic pathways that allow the cell to regenerate ATP.
Anabolic reactions are the opposite of catabolic ones, that is they create newbiomolecules. These include small molecules and building blocks that are notsufficiently available in the food, and macromolecules, in particular proteinsand nucleic acids. Apart from building blocks and ATP, anabolic reactionsalso require a good deal of reducing power in the form of NADPH, and we willaccordingly look at the major pathway devoted to its production, which is thehexose monophosphate shunt (chapter 9).
1.2 Catabolic and anabolic reactions 3
Fructose 1-P
Glucose 6-P
Glucose 1-P
Glycogen
Fructose 6-P
Fructose 1,6-bis-P
Glyceraldehyde 3-P/Dihydroxyacetone-P
1,3-bis-P-glycerate
3-P-glycerate
2-P-glycerate
Phosphoenolpyruvate (PEP)
Pyruvate
Acetyl-CoACO2
Citrate
Isocitrate
α-Ketoglutarate
Succinyl-CoASuccinate
Fumarate
Malate
Oxaloacetate
CO2
CO2
H2
H2O
O2ADP + Pi
ATP
Fructose
Sucrose
Glucose
Amylose
Glyceraldehyde
UDP-GlucoseUDP-Galactose
Galactose 1-PGalactose
Lactose
6-P-Gluconate Ribulose 5-P
Ribose 5-P
Xylulose 5-PSedoheptulose 7-P
Erythrose 4-P
Arginine
Citrulline
ArgininosuccinateOrnithine
Carbamoyl-P
NH3CO2
Urea
Asp
Acetoacetate
β-Hydroxybutyrate
LeuLysPheTrpTyr
PheTyr
Glu
Gln
ArgHisPro
Methylmalonyl-CoA
Propionyl-CoAIleMetThrVal
Odd-numberedfatty acids
Fatty acyl-CoA
Malonyl-CoA
Triacylglycerol
Fatty acids
Glycerol-P Glycerol
HMG-CoA
Cholesterol
CO2
AlaCysSerGly
Figure 1.2 Metabolic pathways covered in this class. Solid arrows indicate singleenzyme reactions, with the exception of the respiratory chain, which converts H2 toH2O. Dashed arrows represent sequences of enzyme reactions.
4 1 Introduction
The relationships described above between catabolic and anabolic pathwaysare summarized in Figure 1.1.
1.3 Metabolic diversity
There are several mainstream metabolic pathways that occur in a wide varietyof living organisms. A good example is glycolysis, the main pathway of glucosedegradation. On the other hand, it is important to note that metabolic processesin organisms other than man or animals may be quite different. Examples are:
1. Photosynthesis, which ultimately enables plants to create all their carboncompounds from CO2 and water. However, even plants do perform catabolicreactions on endogenous macromolecules. An example is the degradation ofstarch, which is stored in large amounts in plant seeds (e.g. wheat and rice) andbulbs (e.g. potatoes). The pathways of starch utilization employed by plantsare entirely analogous to those found in animals.
2. Nitrogen fixation by the bacterium Rhizobium meliloti and related species.Most organisms require nitrogen in reduced form (as ammonia or as part ofamino acids), but Rhizobium is able to convert athmospheric nitrogen (N2) toammonia. This is a process of fundamental importance in agriculture (and infact, to life as such), since the ammonia is required by plants for amino acidsynthesis.
3. Some bacteria that live in quite exotic environments have developed corre-spondingly exotic metabolic pathways. Some of these are capable of extractingenergy from the oxidation of iron or the reduction of sulfur.
While these pathways are certainly very interesting, we will not deal withthem in this class. Instead, we will look at only those metabolic pathways thatoccur in human cells, and not even at all of these. The focus will be on pathwaysthat supply the cell with ATP and reducing power for its various tasks, thatis on catabolic pathways. A summary of the pathways covered in this class isgiven in Figure 1.2. In addition, we will relate some of these pathways to humanhealth and disease.
1.4 Types of foodstuffs
Catabolism starts with foodstuffs. The three major categories of foodstuffsrelevant to human metabolism are named on every box of cereal or cup ofyoghurt (Figure 1.3a). If you look at the top of Figure 1.2, you will see lots ofnames ending in -ose. These are sugars. Glucose, fructose, and galactose aresingle sugar molecules, or monosaccharides. Sucrose und lactose are dimericsugars or disaccharides, whereas amylose is polymeric, or a polysaccharide.Utilization of disaccharides and oligosaccharides starts with their cleavage tomonosaccharides.
1.5 The digestive tract 5
The sum formulas of sugars can approximately be written as (CH2O)n, thatis they formally are multiples of carbon (C) plus water (H2O). They are thereforecollectively referred to as carbohydrates. These form an important part of ourdiet. Indeed, in the typical diets of many countries, the most calories by far arederived from carbohydrates, contained for example in rice, wheat, or potatoes.
To the right of the center in Figure 1.2 you will find triacylglycerol. This isanother major component in our diet: Fat. The decomposition of fat yields fattyacids as the main component; these are further degraded in the β-oxidationpathway to acetyl-CoA. Acetyl-CoA is also an intermediate in the completedegradation of carbohydrates and of proteins. Therefore, acetyl-CoA is a cen-tral hub in metabolism, through which all substrates destined for completeoxidative degradation must pass. Acetyl-CoA is also a precursor in severalbiosynthetic pathways (Figure 1.3b).
The third major type of foodstuff are proteins. Of the twenty standardamino acids found in proteins, we can synthesize only ten ourselves; the otherones are called essential amino acids. Therefore, while a low amount of dietarycarbohydrates or fat can be compensated by our own biosynthesis,1 lack ofdietary protein cannot. Accorgingly, in poor countries, lack of dietary proteinis the most common form of malnutrition.
One class of macromolecules that is not covered here are the nucleic acids.Nevertheless, nucleic acids make up a sizeable fraction of our food. Only thesugar part of each nucleotide—ribose or deoxyribose—can be utilized to gainenergy. The bases—A,C,G,T—can be used as building blocks for the synthe-sis of new nucleotides and nucleic acids. If they are present in excess overbiosynthetic demands, they are modified and excreted.
1.5 The digestive tract
Where does metabolism occur? The first step, the depolymerization of food-stuff macromolecules, occurs extracellularly, certainly in humans but typicallyeven with organisms as simple as bacteria. Depolymerization is accomplishedby digestive enzymes, which are secreted by the cell, and uptake of substratesinto the cell only occurs at the stage of the monomeric breakdown products.Why is that so? Extracellular digestion makes the substrates available to ev-eryone, not just to the cell that has provided the necessary enzymes. It istherefore a potentially ineffective process, especially with unicellular organ-isms that often will find themselves competing for substrates with numerousother species.
1The traditional diet of eskimos, for example, is extremely low in carbohydrates, since itconsists mostly of meat and fish.
6 1 Introduction
Sugars Fat Amino acids
Fat Cholesterol Ketone bodies
Acetyl-CoA CO2+ H2O
ADP ATP
Sugars Fat Amino acids
Fat Cholesterol Ketone bodies
Acetyl-CoAAcetyl-CoA CO2+ H2O
ADP ATP
a)
b)
Foodstuffs
Carbohydrates
Fat
Protein
Sugars
Fatty acids, glycerol
Amino acids
CO2 + H2O
CO2 + H2O
CO2 + H2O
Urea, sulfate
Figure 1.3 Summary of catabolism of the three major classes of foodstuffs. a: Allthree types can be completely broken down to carbon dioxide and water to yield ATPand / or NADPH (see Figure 1.1, right). Amino acids, which contain nitrogen and insome cases sulfur, also yield urea and sulfate when completely degraded. b: Acetyl-CoA is a central intermediate in the breakdown of all classes of foodstuffs. It is also aprecursor in the synthesis of fat, cholesterol, and ketone bodies.
An obvious answer is that there are no transport mechanisms for the uptakeof macromolules across the cell wall. While that is true,2 there is a deeperreason – taking up macromolecules in a non-specific way would open the doorfor all kinds of Trojan horses. Extracellular digestion is a kind of a firewallto exclude hazardous biomolecules.3 Extracellular depolymerization is alsothe strategy employed by our own digestive tract (Figure 1.4). The digestive
2But not universally – for example, bacterial cells have evolved mechanisms for the activeuptake of DNA, which they switch on under special circumstances. This is exploited in the lab intransformation experiments.
3Amoebae, for example, do indeed ingest not only macromolecules but even whole bacteria.They can do so because they confine the ingested material within phagosomes, which they swiftlyflood with acid and aggressive chemicals and enzymes to kill and degrade the bacteria. The sameoccurs in our phagocytes, which are an essential part of our immune system.
1.5 The digestive tract 7
stomach
liver
small
intestine
large
intestine
a)
pancreas
b)
liver
stomachbile
bladder
pancreas
duodenum
bile
duct
pancreatic duct
Figure 1.4 Schematic of the digestive tract. a: Overview. b: The liver and the pancreasboth have secretory ducts that discharge into the duodenum, which is the topmost partof the small intestine.
tract contains specialized organs and cells for the secretion of depolymerizingenzymes, for the performance of the digestion, and finally the uptake of thereleased substrates.
1.5.1 The stomach
The first major section of the digestive tract is the stomach. The stomachcontains gastric acid, which is hydrochloric acid (HCl) with a pH of ~2, whichof course creates a very aggressive environment. The gastric acid has twoimportant biological effects:
1. It sterilizes the food, that is it kills most of the ingested microorganisms.Individuals with impaired secretion of gastric acid or those who take drugsthat inhibit acid secretion, which is necessary in the treatment of gastric orduodenal ulcers, are more susceptible to infectious diseases such as choleraand intestinal tuberculosis.
2. It denatures (or unfolds) proteins. In denatured form, proteins are muchmore accessible to digestion. Protein digestion is initiated right away in thestomach by the protease pepsin. The peptides generated will no longer refold,even though the pH returns to near neutral conditions in the small intestine,so that digestion can be completed by the proteases and peptidases foundthere (Figure 1.5). Most, but not all proteins will be unfolded by gastric acid;an important exception is pepsin itself. The coat proteins of many pathogenicviruses, for example poliovirus, are fairly resistant to gastric acid as well, so
8 1 Introduction
pH 2
Pepsin
pH 7.5
Trypsin etc.
Uptake of small peptides and free amino acids
Figure 1.5 Digestion of proteins. The acidic pH in the stomach causes the proteinsto unfold, which makes the peptide bonds accessible to pepsin. The fragments will notbe able to refold, even though the pH reverts to neutral in the small intestine, so thatthe digestion can be completed, and the final products be taken up.
that these viruses are able to traverse the stomach and then infect the intestinalmucosa.
1.5.2 The pancreas
While the stomach protects the lower parts of the intestinal tract from infec-tious agents and conditions the food for further digestion, most of the hardwork is done in the small intestine. For the digestion to occur, we need specificenzymes for each of the major polymers that occur in the food:
1. Amylases and disaccharidases degrade starch (Figure 1.6) and other car-bohydrates.
2. Proteases and peptidases degrade proteins and release amino acids.3. Lipases digest fat to release glycerol and fatty acids.4. Nucleases release nucleotides from nucleic acids.Most of these digestive enzymes are synthesized by the pancreas. This is
a large gland situated right next to the topmost part of the small intestine,the duodenum, into which it discharges its secretions. Accordingly, patientswith a lack of pancreas function are deficient in the digestion of all kinds offoodstuffs.
Apart from the degradative enzymes, the pancreatic juice contains sodiumbicarbonate. The bicarbonate serves to promptly neutralize the gastric acidupon entering into the duodenum. In keeping with the near-neutral pH thatprevails in the intestine, all the pancreatic enzymes have a roughly neutral pHoptimum, which contrasts with the acidic pH optimum of pepsin.
1.5 The digestive tract 9
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
OH
O
OOH
H
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2OH
O
O
OH
OH
CH2
……
Amylopectin
O
O
OH
OH
CH2OH
O
OH
OH
CH2OH
OH OH
Maltose
α-D-Glucose β-D-GlucoseD-Glucose
CHO
OH
OH
OH
OH
OH
H
H
H
H
H
H
O
OH
OH
CH2OH
OH
OHO
OH
OH
CH2OH
OH OH
branch
Figure 1.6 Structures of glucose, amylose and maltose. Top: Glucose occurs in twoanomeric ring forms, as well in an open chain form. Middle: Amylopectin is a branchedpolymer of α-glucose. The unbranched polymer is called amylose; both are containedin starch. Bottom: Maltose is the disaccharide which results from the degradation ofamylose by amylase. It is in turn cleaved to glucose by maltase at the surface of theintestinal mucosa.
1.5.3 The liver
Among its many other functions, the liver also serves as an exocrine gland.The digestive juice secreted by the liver is known as the bile and is rich in bileacids (see Figure 11.1), which are important in solubilizing fat so as to render it
10 1 Introduction
accessible to enzymatic cleavage.4 The bile may be stored and concentrated inthe gall bladder, or directly secreted together with the pancreatic juice (the bileduct and the pancreatic duct join immediately before reaching the duodenum).Like the pancreatic juice, the bile is rich in sodium bicarbonate.
In keeping with the occurrence in the bile of bile acids but not of digestiveenzymes, patients suffering from a disruption of bile secretion will show adeficiency in the digestion of fat but not of proteins or carbohydrates. Apartfrom the secretion of bile, the liver has a key role in processing substrates afteruptake from the intestine. This is discussed in section 1.5.6.
1.5.4 The small intestine
After digestion, the metabolites have to be taken up by the cells at the sur-face of the mucosa of the small intestine, which comprises the duodenum,the jejunum, and the ileumsmall intestinesections of. How does this uptakework? Most nutrients are taken up by active transport, which can transportsolutes energetically uphill, that is against their concentration gradients. Activetransport is necessary for the quantitative uptake of the nutrients. An impor-tant example is the transport of glucose, the most important end product ofcarbohydrate digestion. The uptake of glucose is driven by the simultaneousuptake of two sodium ions per molecule of glucose; this coupling is effectedby the SGLT1 transporter (Figure 1.7). While sodium is plentiful in the gutlumen, its concentration is low inside the cells. The uphill transport of glucoseis therefore driven by the simultaneous downhill movement of sodium. Similartransporters exist for other sugars (e.g. galactose) and for amino acids.
The amount of nutrient substrates that have to pass across the surface ofthe gut is considerable; therefore, a large active surface is required. You haveprobably heard that the length of the small intestine is likewise considerable –about twice the height of your body. In addition, the inner surface of the smallintestine is folded, and the folds in turn have a shaggy surface, as have theindividual cells. All these factors combine to maximize the effective surfaceavailable for substrate uptake. This is illustrated in Figure 1.8.
1.5.5 The lower small intestine and the large intestine
All digestion and nutrient absorption occurs in the small intestine, mostly in itsupper portions. In fact, even the lower segments of the small intestine usuallydon’t get to do much work in nutrient uptake but have a more specialized rolein the reuptake of bile acids, and of things such as vitamin B12, the lack ofwhich causes pernicious anemia. In the large intestine, essentially no nutrientsare left for absorption. What, then, is the function of the large intestine? A
4Bile acids solubilize fat because they are detergents, and as such they are also useful forremoving tough stains from your laundry.
1.5 The digestive tract 11
GlucoseGlucose Glucose
2 Na+ 2 Na+
SGLT1 GLUT2
gut lumen bloodcytosol
Figure 1.7 Mechanism of glucose uptake from the gut lumen. The transport againstthe concentration gradient is powered by cotransport of sodium ions via the trans-porter SGLT1 (SGLT=sodium glucose transporter), while the transport into the blood isdown a concentration gradient and can therefore be accomplished by simple facilitateddiffusion through GLUT2 (GLUT=glucose transporter).
key aspect of the large intestine is that it is inhabited by the so-called gut flora,which comprises literally trillions of bacteria.5 What are these guys doing there,other than producing foul smells, and why do we afford them such generousshelter? There are several benefits of this:
1. Some bacteria are capable of synthesizing some vitamins, such as folicacid, the lack of which again causes anemia, with symptoms and laboratoryfindings very similar to those observed when vitamin B12 is lacking. Both folicacid and vitamin B12 are required in the synthesis of nucleic acids. Inhibitionof DNA synthesis has the strongest impact in rapidly proliferating tissues suchas the bone marrow, which is the site of blood cell regeneration.
2. Our diet contains some substrates such as strange sugars and polysac-charides that we cannot utilize or absorb, and which therefore travel throughthe small intestine unaltered. Some bacteria can break down these substrates.6
Since the milieu in the colon is anaerobic—that is, it lacks oxygen—breakdownwill not be complete. Fairly common end products of fermentation are smallacids such as acetic or propionic acid7 Some of these products are then takenup across the colon epithelium and utilized in our metabolism.
5In fact, the number of bacteria in our large intestine is higher than that of our own body cells!So, arguably, we are in fact prokaryotic ;)
6Testing for utilization of all kind of sugars—rhamnose, raffinose, melibiose and so forth—aswell as amino acids is in fact a traditional methodology for classifying enteric bacteria.
7Our good old friend Escherichia coli and other bacteria also produce formic acid, which theythen cleave to H2 and CO2, which are in turn released as gases.
12 1 Introduction
blood, lymphatic vessels
microvilli
villi
Figure 1.8 Structure of the mucosa of the small intestine. Top: Schematic, show-ing the surface structure at increasing power of magnification. The inner sur-face of the intestine is folded, and the folds in turn have a shaggy appearancebecause the mucosa is folded up again into villi. The epithelial cells that consti-tute the mucosal surface are in turn covered by microvilli. Bottom: Villi, shownin a stained section of the intestinal mucosa (light microscopy, left), and microvillion the surface of an epithelial cell (scanning electron microscopy). Below the sur-face, you can see the folded membranes of the endoplasmic reticulum. The mi-croscopic images have been reproduced, with permission from Roger Wagner, fromhttp://www.udel.edu/Biology/Wags/histopage/histopage.htm.
3. The breakdown of non-utilizable substrates in bacterial metabolism alsohelps us to retrieve almost all of the water from the gut lumen. The overallamount of fluid secreted into the digestive tract is about 7 liters per day. At
1.5 The digestive tract 13
Liver
Vena portae and tributaries Liver artery
Liver vein
Systemic
circulation
Figure 1.9 The portal circulation. The intestines receive arterial blood from the heart,just like any other organ. The venous blood, however, is not passed back directly tothe right heart, as is the case with practically all other organs, but first reaches the liverthrough the portal vein. The liver also receives a direct supply of oxygen-rich bloodthrough the liver artery. After passage through the liver, the blood received throughboth the portal vein and the liver artery is fed into the general circulation.
the end of the small intestine, 5-5.5 liters have been reclaimed. Recovery ofthis residual amount is hampered by the fact that the non-utilized solutes areosmotically active, that is they will hold on to water and drag it down the pipe.Utilization of these solutes in bacterial metabolism reduces the osmotic activityof the colon content, enabling a more complete reuptake of water.8
So, you see that our intestinal bacteria are actually quite useful. On theother hand, the bacterial fermentation also produces ammonia and other toxicproducts that must be captured and disposed of by the liver. This is not aproblem in healthy individuals but can become one in persons suffering fromliver disease.
1.5.6 The portal circulation
Upon uptake, most solutes will be exported on the other side of the mucosalcells and then find themselves in the blood stream. A peculiarity of the in-testines consists in the fact that all blood drained from them is first passed
8On the other hand, increasing the osmotic activity of the gut content is a straightforwardway of limiting water reuptake in order to treat constipation. A traditional drug based on thisprinciple is sodium sulfate (now out of fashion).
14 1 Introduction
on to the liver via the portal vein (Figure 1.9) portal circulation before beingreleased into the general circulation. This serves a twofold purpose:
1. It gives the liver a chance to take excess amounts of substrates—glucose,amino acids—out of circulation and to store and process them. This servesto maintain stable blood nutrient concentrations, which is important for thewell-being of the more sensitive and fastidious cells in the rest of the body.
2. The bacteria in the large intestine produce ammonia and other toxicmetabolites, which are cleared by the liver. In patients with liver failure, thesetoxic metabolites spill over into the systemic circulation, which will amongother things lead to disturbances of cerebral function. This activity of the liveralso affects many drugs; the inactivation of drugs by the liver immediatelyfollowing intestinal uptake is known as the first pass effect.9
The liver has a particular tissue structure that enables it to exchange soluteswith the blood very efficiently. While in most organs the blood is contained inblood vessels with clearly defined boundaries and walls, the liver has a sponge-like structure that permits direct contact of the blood plasma with the livercells (Figure 1.10).
1.6 What’s next?
Digestion is only a preparatory step in metabolism. The really interesting partstarts once the substrates have been absorbed and made their way to the liver.Their fate there will depend on the prevailing metabolic situation. The utiliza-tion of glucose, the most important single substrate in energy metabolism, iscontrolled by the hormones insulin and glucagon, both of which are secretedin the endocrine islets of the pancreas:
1. If glucose is plentiful, typically after a meal, a significant fraction will beretained in the liver and stored there in the form of glycogen, which is a polymerclosely similar to amylopectin. The stored glycogen is broken down againto glucose, which stabilizes blood glucose levels during prolonged intervalsbetween meals. Once the glycogen stores are stocked up to the roof, glucoseand amino acids will be turned into fat, which will then be forwarded to theperipheral fat tissue for storage.
2. If glucose is available but in high demand due to exertion, the liver willpass on the glucose to the periphery for utilization.
3. If it is scarce, the liver will use part of the amino acids it may receivefrom the intestine to turn them into glucose, which it then passes on to theperiphery.
Dietary fat is processed differently from water-soluble substrates, that issugars and amino acids. It is packaged into lipoproteins directly in the intestine,
9The first pass effect, and pharmacokinetics in general, are discussed in more detail in myBiochemical Pharmacology course notes.
1.6 What’s next? 15
To liver
vein
Portal vein branch (from intestine)
Liver artery branch
a)
b)
Figure 1.10 Blood circulation and tissue perfusion in the liver. a: The liver tissuehas a honeycomb structure; each hexagon constitutes one liver lobule. The liver arteryand portal vein branches are located at the corners of each lobule. The blood seepsout of the artery and portal vein branches, into the sinusoids of the liver lobule. Itis collected by the lobule’s central vein, which drains it toward the liver vein and thegeneral circulation. b: Higher power view, showing the sponge-like structure of theliver tissue. The liver cells are stained in brown; the white space in between are thesinusoids. While in the sinusoids, the blood gains surrounds virtually every liver cell.The microscopic images have been reproduced, with permission from Roger Wagner,from http://www.udel.edu/Biology/Wags/histopage/histopage.htm.
and it bypasses the liver because it is delivered into the lymphatic vessels ratherthan into the blood stream.
We will consider all these processes in turn. At the end of this class, we willnot only have gained a solid grasp of these pathways, but we will also have thetools to understand in depth what goes wrong in metabolic diseases such asphenylketonuria, lactose intolerance, and diabetes mellitus.
Chapter 2
Refresher
This chapter attempts to summarize some essential concepts from second yearbiochemistry. Feel free to skip it if you remember a thing or two from thatdistant past.
2.1 How enzymes work: Active sites and catalytic mecha-nisms
Open any textbook of biochemistry and you will be presented with an over-whelming number of figures depicting the crystal structures of a multitude ofenzymes. Of course, the three-dimensional structures of enzymes are crucialto their functions. Many enzymes are just regular protein molecules, composedof nothing else than the 20 standard amino acids. If you mix of all these aminoacids in free form at, say, 1 mM each, this mixture will not have any significantcatalytic activity. This suffices to show that it is the precise arrangement of theamino acids in the enzyme molecule that brings about its specific function.
Since the α-amino and α-carboxyl groups of the amino acids are hooked upto each other in the polypeptide chain, they usually do not directly contributemuch to the catalytic effect of the enzyme. Instead, it is the side chains that aredirectly engaged win the reaction.1 A very good example of this is chymotrypsin(Figure 2.1). Chymotrypsin is one of the major proteases in the human digestive
16
2.1 How enzymes work: Active sites and catalytic mechanisms 17
NH
O
R4
NH2
O
R1
NH
O
R2
NH
O
R3
NH
C
O
R2
NH
O
R3
R
R
OHOH
NH
C
O
R2
NH
O
R3
R
R
O
H
H
NH
C O
R2
R
OH
N
O
R3
RH
H
OH
NH
C
O
R2
NH
O
R3
R
R
O
Ser−Enzyme
O
NH
C
O
R2
NH
O
R3
R
R
Ser−Enzyme
A
H
NH
CO
R2
R
O
Ser−Enzyme
N
O
R3
RH
H
A−
Ser 195
Asp 102
His 57
Ser 195
Asp 102
His 57
NH2
O
OO
NH2
O
N
N
H
NH2
O
O
H
NH2
O
OOH
NH2
O
N
N
H
NH2
O
O
Attack onsubstratepeptide bond
Asp 102
His 57
Ser 195
a)
b)
c)
d)
Figure 2.1 Structure and mechanism of chymotrypsin. a: The peptide bond cleavedby chymotrypsin. b: The interplay of the side chains of histidine 57, aspartate 102, andserine 195 that results in the deprotonation of serine. All three side chains are close toeach other in the active site of the enzyme. c: The enzyme- catalized reaction. d: Thebase-catalyzed reaction for comparison.
tract, where its job is to knock down large protein molecules into small peptidesthat are then further processed by peptidases.
1You will note that, nevertheless, many textbook figures give the enzyme structures as ribbondiagrams that just show the fold of the protein backbone but omit the side chains. Therefore,these diagrams are completely useless for understanding the catalytic mechanism of an enzyme.
18 2 Refresher
How does chymotrypsin do that? The enzyme-catalyzed reaction (Figure2.1c) is similar to alkaline hydrolysis (Figure 2.1d). In alkaline hydrolysis, ahydroxide ion, which is a strong nucleophile, attacks the carbon in the peptidebond that carries a partial positive charge. In the enzyme-catalyzed reaction, adeprotonated serine residue of the enzyme (Ser 95) plays a role similar to thatof the hydroxide ion.
Now, you know that serine normally is a neutral amino acid – its -OH groupdoes not spontaneously dissociate, no more than the -OH group of alcoholdoes.2 The question therefore is, how does the enzyme deprotonate its ownserine side chain? This is brought about by placing the serine next to a histidineand an aspartate inside the active center (Figure 2.1). Aspartate deprotonateshistidine, which in turn deprotonates the serine residue. This motive—asp, his,ser—is very widespread among proteases and esterases, so much so that it iscommonly referred to as the catalytic triad. For example, the protease trypsinand several lipases that occur in human metabolism have this motif and sharethe same mechanism of catalysis.3
While with many enzymes the protein molecule and its amino acid sidechains are sufficient for catalysis, many others require coenzymes for theircatalytic activity. Very often, both active-site amino acid side chains and coen-zymes are required. For example, amino acid transaminases (see Figure 12.4)have a molecule of the coenzyme pyridoxalphosphate bound to the active site,which cooperates with an active site lysine during the enzyme reaction.
Most enzymes have just one active site, or if they are multimeric one activesite per subunit. However, there are exceptions: Fatty acid synthase (see section10.5) has as many as eight different active sites on each subunit. Multi-enzymecomplexes such as pyruvate dehydrogenase (see section 5.1) have one activesite per subunit but combine different types of subunits and enzyme activitiesin one functional assembly.
2.2 Classification of Enzymes and enzyme reactions
When looking at enzyme names such as transketolase or phosphorylase, youwill note that these names don’t tell you what reactions exactly the enzymesmay catalyze, or what substrates they operate upon. A complete descriptionshould mention the coenzymes required, the substrates and the particularbonds in the substrates that are being severed or created. A nomenclaturethat meets these criteria has been developed by the Enzyme Commission ofthe IUBMB (International Union of Biochemistry and Molecular Biology). In theIUBMB nomenclature, the enzyme transketolase bears the formidable name:
2Otherwise, beer would taste sour – imagine how rotten that would be.3Variations of this motif, for example in the proteasome, may contain glutamic acid instead of
aspartic acid, or threonine instead of serine. These variations still contain the same functionalgroups and work the same way.
2.3 Energetics of enzyme-catalyzed reactions 19
Sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetrans-ferase (no, I can’t remember it either).
Such names, of course, are rather lengthy, and their use is not very wide-spread. To make the tasks of tracking and bookkeeping more manageable,these names are supplemented with numeric codes. In the IUBMB scheme,enzymes are put into one out of six classes according to the reactions theycatalyze. These classes are:
1. Oxidoreductases. These catalyze redox reactions, frequently involvingone of the coenzymes NAD+, NADP+, or FAD.
2. Transferases. These bring about the transfer of functional groups, for ex-ample a phosphate group from ATP to another metabolite, which activatesthe latter and sets it up for subsequent reaction steps.
3. Hydrolases. These catalyze hydrolysis reactions, such as those involvedin digestion of foodstuffs.
4. Lyases. These enzymes effect elimination reactions that result in theformation of double bonds.
5. Isomerases. These facilitate the interconversion of isomers. We will meettwo examples as soon as we get into glycolysis.
6. Ligases, which form new covalent bonds at the expense of ATP hydrolysis.
Of course, within each of these main classes, there are subclasses and sub-subclasses that correspond to details of substrates and mechanisms of the enzymereactions. Each individual enzyme activity is assigned an individual numberwithin a sub-sub class, so that we wind up with a four-figure designation, whichis preceded by the letters EC (Enzyme Commission). The website http://www.chem.qmul.ac.uk/iubmb/ gives a list of all the enzyme activities recorded bythe IUBMB classification.
One good thing about this classification is that it is rarely used. The “rec-ommended names”, which most of the time happen to be the traditional ones,are used instead. The other good thing is that it has a sound appreciation ofpriorities. EC 1.1.1.1 is the single most important enzyme in student lifestyle –namely, alcohol dehydrogenase, or, as IUBMB puts it, alcohol:NAD oxidoreduc-tase. This laudable enzyme, residing in the liver, degrades ethanol, and withoutit we would be drunk all the time!
2.3 Energetics of enzyme-catalyzed reactions
With each enzymatic reaction, as with any other chemical reaction, energycomes in with two questions: (1) Will the reaction proceed at all in the desireddirection? (2) If it does, will it proceed at a sufficient rate?
The first question is decided by the free energy of the reaction, ∆G, whichwhen negative will make the reaction go forward. The second question dependson the activation energy, ∆G∗, which forms a barrier between the two states of
20 2 Refresher
Barrage
Turbine
Figure 2.2 The Kochelsee-Walchensee hydroelectric power system. The Walchenseeis situated above the Kochelsee. Artificial conduits connect the two lakes and also drainother lakes into the Walchensee.
the reactants. The very short-lived, energy-rich state at the top of this barrier iscalled the transition state. Enzymes can make a big difference to the activationenergy ∆G∗ and thus accelerate reactions, but they cannot change the freeenergy ∆G—and therefore, the direction—of the reaction.
2.3.1 A simile
The different roles of ∆G and ∆G∗ in biochemical reactions can be illustratedwith a simile. Figure 2.2 below shows two lakes in the German Alps. TheWalchensee4 is situated 200 m above the Kochelsee. A conduit was dug acrossthe barrier between these two lakes to make the water flow downhill and drivea hydroelectric turbine. Additional tunnels drain other lakes and rivers toenhance the supply of water to the Walchensee.
Driven by the considerable hydrostatic pressure accruing during the flowdownhill, this system generates quite a bit of electrical energy. It can be likenedto a metabolic pathway:
• The difference in altitude between the reservoirs is similar to the freeenergy difference, ∆G, between two metabolites.
• The height of the barrier between the two lakes is similar to the activationenergy (∆G∗) of the transition between two metabolites. Enzymes facili-tate the interconversion of metabolites by creating “tunnels” that bypassthe activation energy barrier between them.
• In the hydro-electric system, tunnels can be opened or shut by barrages toaccommodate different amounts of rainfall or of electrical energy required.Likewise, enzymes have switches to regulate their activity to allow foradjustments in the flow along metabolic pathways.
4Der See=German for the lake, die See=German for the sea
2.4 The role of ATP in enzyme-catalyzed reactions 21
• Like the conduits, enzymes can facilitate the flow and (typically) controlits rate but not its direction. The direction of the flow will depend solelyon the difference in altitude (∆G).
It is usually best not to take analogies too far, because otherwise they be-come obfuscating rather than enlightening. Here are some dissimilarities:• All tunnels are basically alike. In contrast, each enzyme needs a specific
“trick” or catalytic mechanism suited to the specific metabolites it is deal-ing with. Investigating the catalytic mechanisms of individual enzymes isan important part of biochemistry.
• The energy level of a pool in the hydro-electric system is completelydescribed by its altitude. However, the free energy of a metabolite isdependent on its concentration; the lower the concentration, the lowerthe free energy.
• Although enzymes cannot revert an endergonic reaction (that is, a reactionwith ∆G > 0) in isolation, they can make it go uphill by coupling it toanother one that is exergonic, so that the overall ∆G becomes negative.
• Finally, water will never spontaneously flow uphill – molecules, however,do occasionally move spontaneously to somewhat higher energy levels.
If molecules are offered two states with different energy levels, such as twodifferent conformations or two different tautomeric states, they will sponta-neously distribute between them and establish equilibrium. In equilibrium,the relative occupancy of these two states will solely depend on the energydifference between them:
n1
n2= e−
∆GRT (2.1)
where n1 and n2 represent the numbers of molecules in the high and lowenergy states, respectively. (R is the gas constant, whereas T is the absolutetemperature.)
The above equation applies to the distribution of the initial and the finalstates of a reaction. It also applies to the distribution between molecules be-tween the initial state and the transition state of a reaction. The tendency ofmolecules to spontaneously assume states of higher energy explains that chem-ical reactions can occur at all, even though the energy level of the transitionstate is always higher than those of the initial and final states. However, thehigher the activation energy, the more rarified the transition state will become.The number of molecules that can make it across the barrier thus becomessmaller, and the reaction slower with increasing activation energy.
2.4 The role of ATP in enzyme-catalyzed reactions
The most common exergonic reaction that is utilized by enzymes to drive en-dergonic ones is the hydrolysis of the phosphodiester bonds in ATP. While this
22 2 Refresher
CHNH2
CH2
CH2
OOH
O NH2
OPO
O
O
PO
O
O
Adenosine
O P O
O
O
+
+
CHNH2
CH2
CH2
OOH
O O P O
O
O
OPO
O
O
PO
O
O
Adenosine
NH3
+
CHNH2
CH2
CH2
OO
O O
OPO
O
O
PO
O
O
PO
O
O
Adenosine+
Figure 2.3 The catalytic mechanism of glutamine synthetase. The terminal phos-phate is first transferred to glutamate to form a carboxyphosphate. In this energy-richintermediate, the phosphate is a good leaving group and is easily substituted by am-monia.
is a general principle in enzymology, it is important to understand that there isno equally general chemical mechanism of ATP utilization: Each enzyme needsto find its own way of actually linking it to the reaction it needs to drive. As anexample, here is how glutamine synthetase does it. This enzyme uses ATP toproduce glutamine from glutamate and ammonia (Figure 2.3).
While the net turnover of ATP is hydrolysis, the phosphate group is actuallyfirst transferred to the substrate to create an intermediate product, glutamate-5-phosphate. The phosphate group in this compound—a mixed anhydride—isa very good leaving group, which facilitates the subsequent substitution by
2.5 Regulation of enzyme activity 23
Fructose-6-PATP (cosubstrate)
ADP (regulator)
Figure 2.4 Structure of the enzyme phosphofructokinase (shown in wire-frame) withbound substrate (fructose-6-phosphate), cosubstrate (ATP) and allosteric regulator(ADP), all shown in space-filling mode. Substrate and cosubstrate reside in the activesite, while ADP binds to a separate regulatory site.
ammonia. Therefore, the utilization of ATP is an integral part of the reactionmechanism of this enzyme. We will see additional examples of ATP usage inenzyme catalysis in the remainder of this course.
2.5 Regulation of enzyme activity
The enzyme phosphofructokinase catalyses the following reaction:
Fructose-6-phosphate + ATP→ Fructose-1,6-bisphosphate + ADP (2.2)
This reaction occurs as an early step in the degradation of glucose, whichultimately serves to replenish ATP from ADP and phosphate. It therefore makessense that phosphofructokinase should be stimulated by ADP.
To accomplish this stimulation, ADP binds to a distinct site on the enzymethat is far away from the active site; it therefore clearly does not directlyparticipate in the reaction (Figure 2.4). Instead, the binding of ADP changesthe conformation of the entire enzyme molecule. The change will also affectthe active site and enhance the efficiency of catalysis there. This mode ofaction is known as allosteric regulation and is exceedingly common. Allosteric
24 2 Refresher
Inactive
conformation
Allosteric
inhibition
Active
conformation
R A
Allosteric
activation
R A
a)
b)
Inhibition by phosphorylation
P
c)
d)
Allosteric regulation and cooperativity
Figure 2.5 Enzyme regulation by allosteric effectors and by phosphorylation. a: Theregulatory binding site for the allosteric effector (R) is distinct from the catalytic activesite (A) of the enzyme. The enzyme can assume two distinct conformations, and boththe regulatory site and the active site are affected by the transition between them. Theenzyme is active only in one of these conformations. b: An allosteric inhibitor bindsto a regulatory site in the inactive conformation; the energy of binding favours thisconformation. An allosteric activator binds and stabilizes the active conformation.c: Most allosteric enzymes are multimeric. The conformational transition affects theinterfaces between the subunits, thereby enforcing the transitions of all subunits tooccur synchronously or cooperatively. d: Regulation by phosphorylation also worksby selective stabilization of one conformation. Like allosteric regulation, it can beinhibitory (as shown here) or stimulatory.
effectors can be either stimulatory, as ADP is in this example, or inhibitory. Asan example of the latter, ATP not only binds to the active site but also acts asan allosteric inhibitor of phosphofructokinase – which again makes sense inthe context of overall physiological regulation.
The workings of allosteric regulation are schematically depicted Figure 2.5.The enzyme has two possible conformations that are in equilibrium with eachother. An allosteric activator will bind selectively to the regulatory site as it oc-curs in the active conformation and thereby shift the equilibrium towards thisconformation. Conversely, an inhibitor would bind selectively to the inactiveconformation and thereby stabilize it. As you can see, activators and inhibitorsmay share the same regulatory site; this is the case in the above example ofphosphofructokinase with ATP and ADP. Note, however, that phosphofructoki-nase has additional allosteric sites that permit regulation by other effectors (seeFigure 7.4). Although it is not theoretically necessary, it seems that all allostericenzymes occur as oligomers. In Figure 2.5c, a dimeric enzyme is shown, whichis not uncommon, but often the number of subunits is considerably higher.Their oligomeric nature enables enzymes to behave cooperatively, that is toreact more sensitively to changes in effector concentration.
2.5 Regulation of enzyme activity 25
Another important means of enzyme regulation is through protein phos-phorylation. This occurs by protein kinases, which transfer a phosphate groupfrom ATP to a specific protein side chain on the regulated enzyme. The mecha-nism of regulation by phosphorylation is not really that different from allostericregulation. The only difference is that in this case the regulator is more stablyattached, so that the regulatory effect will last longer. Like allosteric regulation,it can be inhibitory (as shown in Figure 2.5d) or stimulatory. Many enzymes aresubject to regulation both by allosteric effectors and by phosporylation.
Note that the functional effect to any specific allosteric regulator, and ofphosphorylation as well, depends entirely on the enzyme in question. Forexample, while ATP inhibits phosphofructokinase, it activates the function-ally opposite enzyme fructose-1,6-bisphosphatase (section 7.3.2). Likewise,phosphorylation by protein kinase A inhibits glycogen synthase but activatesglycogen phosphorylase (section 8.4).
While all mechanisms discussed so far modulate the activity of existingenzyme molecules, the overall of an enzyme may also be varied by changingthe abundance of enzyme molecules. Firstly, the transcription of the geneencoding the enzyme in question can be turned on or off. This mechanism isemployed by many hormones, in particular steroid hormones such as cortisoneand by thyroid hormones. Enzyme molecules can also be tagged for acceleratedproteolytic degradation. Similarly, the stability of the enzyme messenger RNAsencoding specific enzymes can be regulated up or down, with correspondingeffects on the abundance of the enzyme molecules. Hormones may affect theactivity of an enzyme at more than one level. For example, insulin increasesthe activity of glycogen synthase by way of transcriptional induction, increasedmRNA stability, and protein phosphorylation.
Chapter 3
Glycolysis
Glucose is a key metabolite in human metabolism, and we will spend a good bitof time on the several pathways that are concerned with the utilization, storage,and regeneration of glucose.
3.1 Overview of glucose metabolism
There are five major pathways of glucose metabolism:1. Glycolysis, which accomplishes the degradation of glucose to pyruvate.
Its main purpose is the generation of energy (ATP). Glycolysis generates someATP directly, and a lot more indirectly through the subsequent oxidation ofpyruvate. The need for ATP is universal, so that the glycolytic pathway is foundin every cell of our body.
2. The hexose monophosphate shunt. This pathway also breaks down glu-cose, but the main product is not ATP but NADPH. NADPH is universally neededas a reducing agent, so that this pathway is ubiquitous, too.
3. Glycogen synthesis. Glycogen is a polymeric storage form of glucose, notunlike starch, which is found in plants. This pathway is quantitatively mostimportant in the liver and striated muscle,1 although some is found in in othertissues also. The glycogen synthesized is retained within the same cell.
1Striated muscle comprises skeletal muscle and heart muscle. The second major type ofmuscle tissue is smooth muscle, which occurs in blood vessels and internal organs and is underautonomous control.
26
3.2 The place of glycolysis in glucose degradation 27
Glucose Pyruvate Acetyl-CoAFatty acids,triacylglycerol
CO2
“H2“
H2O
O2
1 2 3
4
5
ADP + Pi
ATP
H2O
Figure 3.1 Overview of glucose degradation. 1: Glycolysis; 2: Pyruvate dehydroge-nase; 3: Fatty acid synthesis; 4: Citric acid cycle; 5: Respiratory chain. The "H2" that isproduced in the citric acid cycle and oxidized in the respiratory chain is not gaseousbut bound to co-substrates.
4. Breakdown of glycogen to glucose, like glycogen synthesis most impor-tant in liver and muscle. This pathway is activated if the current external supplyof glucose is low, as it is between meals. In the liver, the glucose generatedfrom glycogen is released into the general circulation. Sceletal muscle cellsmay utilize the glucose themselves, for the purpose of ATP synthesis and thenmuscle activity (contraction).
5. Gluconeogenesis. This pathway turns pyruvate derived from amino acidsinto glucose; it thus is essentially the reversal of glycolysis. It, too, is activatedin times of low external glucose supply. The amino acid substrates may be ob-tained from a protein-rich diet—for example if we feast on meat exclusively—orby breaking down internal protein, mainly in skeletal muscle. Gluconeogenesisoccurs in the liver and in the kidneys.
We will consider all these pathways in their turn, starting with glycolysis.
3.2 The place of glycolysis in glucose degradation
Figure 3.1 gives an overview of the steps involved in the complete degradationof glucose. It is evident that, in the overall process, glycolysis is only thefirst step. The pyruvate it generates is turned into acetyl-CoA by pyruvatedehydrogenase. Acetyl-CoA is completely degraded in the citric acid cycle andthe respiratory chain. While some ATP is generated at each of these stages,most of it is produced in the respiratory chain.
If glucose is available in excess of immediate needs and of glycogen storagecapacity, it will still be broken down by glycolysis and pyruvate dehydrogenaseto acetyl-CoA. However, acetyl-CoA will then be fed into fatty acid synthesis,which occurs in the liver and the fat tissue.
28 3 Glycolysis
3.3 Reactions in glycolysis
Glycolysis involves 10 enzymatic reactions, summarized in Figure 3.2:1. The phosphorylation of glucose at position 6 by hexokinase,2. the isomerization of glucose-6-phosphate to fructose-6-phosphate by
phosphohexose isomerase,3. the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate
by phosphofructokinase,4. the cleavage of fructose-1,6-bisphosphate by aldolase. This yields two
different products, dihydroxyacetone phosphate and glyceraldehyde-3-phos-phate,
5. the isomerization of dihydroxyacetone phosphate to a second moleculeof glyceraldehyde phosphate by triose phosphate isomerase,
6. the dehydrogenation and concomitant phosphorylation of glyceralde-hyde-3-phosphate to 1,3-bis-phosphoglycerate by glyceraldehyde-3-phosphatedehydrogenase,
7. the transfer of the 1-phosphate group from 1,3-bis-phosphoglycerate toADP by phosphoglycerate kinase, which yields ATP and 3-phosphoglycerate,
8. the isomerization of 3-phosphoglycerate to 2-phosphoglycerate by phos-phoglycerate mutase,
9. the dehydration of 2-phosphoglycerate to phosphoenolpyruvate by eno-lase, and finally
10. the transfer of the phosphate group from phosphoenolpyruvate to ADPby pyruvate kinase, to yield a second molecule of ATP.
3.4 Mechanisms of enzyme catalysis in glycolysis
Metabolic reactions are catalyzed by enzymes. Enzymes are not magicians butsophisticated catalysts, and their chemical mechanisms are often understoodquite well, at least in principle. We will look at a few selected examples that il-lustrate various mechanisms of catalysis that occur similarly in other metabolicpathways, too.
3.4.1 Hexokinase
Our first example is hexokinase, which carries out the first reaction in the gly-colytic pathway. This type of reaction—the transfer of the terminal phosphategroup from ATP onto a hydroxyl group on the substrate—is a very commonreaction in biochemistry, and we will see many more examples in this class.The mechanism of phosphorylation is always the same, so it suffices to discussit once.
Most reactions that involve the transfer of a phosphate group from ATPto something else are exergonic, that is they are energetically favourable. Forexample, the hydrolysis of ATP—which is the transfer of the phosphate group
3.4 Mechanisms of enzyme catalysis in glycolysis 29
O
OH
OH
OH
OH
OH
Glucose
ATP ADP
1
O
O
OH
OH
OH
OH
P O-
OH
OGlucose-6-P
2
OOH
OH
OH
OH
O
P O-
O
OH
Fructose-6-P
ATP
ADP
3
OO
OH
OH
OH
O
P O-
O
OH P O-
OH
O
Fructose-1,6-bis-P
4
O
O
OH
POH O-
O
OH
O
O
POH O-
O
+
GAD-3-P DHAP
5
NAD+NADH+H+
Pi
6
O
O
OH
POH O-
O
O
P O-
OH
O
1,3-Bisphosphoglycerate
ADP
ATP
7
O-
O
OH
POH O-
O
O
3-Phosphoglycerate
8
O-
OH
O
O
P
O-
OH
O
2-Phosphoglycerate
9
O-
O
O
P
O-
OH
O
Phosphoenolpyruvate
ADP ATP
10
O-
O
O
Pyruvate
Figure 3.2 The glycolytic pathway. Enzymes: 1, Hexokinase; 2: Phosphohexoseisomerase; 3: Phosphofructokinase; 4: Aldolase; 5: Triose phosphate isomerase; 6:Glyceraldehyde-3-phosphate dehydrogenase; 7: Phosphoglycerate kinase; 8: Phospho-glycerate mutase; 9: Enolase; 10: Pyruvate kinase. Reversible reactions are indicatedby double arrows, irreversible ones by single arrows. The reversible reactions are partof both glycolysis and gluconeogenesis; irreversible ones require workarounds in glu-coneogenesis. (Abbreviations: -P, phosphate; GAD-3-P, glyceraldehyde-3-phosphate;DHAP, dihydroxyacetonephosphate)
30 3 Glycolysis
XR P
O
O
O O OP
O
O
OP
O
O
Adenosine
OP
O
O
O OP
O
O
OP
O
O
Adenosine
XR
OP
O
O
O OP
O
O
OP
O
O
Adenosine
Mg++
Lys
NH3
+
Arg
NH
NH3
+
NH
XR
a) b)
Figure 3.3 Mechanism of phosphate group transfer from ATP. a: The phosphorus inthe center of the terminal phosphate group is attacked by a nucleophile, which often isan anion (R–X–) but can also be a free electron pair. Attack is inhibited by the negativecharges on the oxygen atoms around the phosphorus. This electrostatic repulsionis responsible for the high activation energy barrier of the uncatalyzed reaction. b:ATP is activated towards nucleophilic attack through electrostatic shielding both bymagnesium and by positively charged amino acid side chains in the active site of theenzyme.
to water—has a free energy of −35 J/K mol. It is interesting to note then that,despite the abundance of water, ATP is quite stable in solution. This indicatesthat there must be a high activation energy barrier that resists cleavage of thephosphodiester bond.2
This energy barrier is due to the negative charges within the phosphategroup that shield the central phosphorus from nucleophiles that likewise arenegatively charged. Accordingly, to lower this barrier, kinases provide com-pensating positive charges within the active sites, which engage the negativecharges on the ATP molecule and thereby facilitate nucleophilic attack (Figure3.3).
The intracellular concentration of glucose will be in the low millimolar range,whereas water is present at a concentration of more then 30 mol/l. Apart fromthe task of activating ATP, then, hexokinase also must ensure that the activatedATP does indeed react with the hydroxyl group on the glucose molecule butnot of water. How is this specificity accomplished?
Like many other enzymes, hexokinase exists in an open and a closed confor-mation. It binds its substrate and cosubstrate, glucose and ATP, in the openstate, whereupon it changes its conformation to the closed state. This changeis necessary for the enzyme’s catalytic activity, and it also is accompanied bythe expulsion of water from the active site (Figure 3.4). In this way, the onlyhydroxyl group that remains in the vicinity of the activated ATP will be thaton the C6 of glucose. However, if we offer the enzyme xylose as a substrate
2If you are unclear about the difference between free energy and activation energy of a chemicalreaction, have a look at section 2.3.
3.4 Mechanisms of enzyme catalysis in glycolysis 31
O
OH
OH
OH
OH
CH2
OH
O
OH
OH
OH
OH
O H
H
Glucose
Xylose + water
a) b)
Figure 3.4 How hexokinase circumvents ATP hydrolysis. a: After binding of itssubstrate glucose (red/spacefill) and its cosubstrate ATP (not shown), hexokinase(blue/wire- frame) adopts a closed conformation, in which substrate and cosubstrateare buried within the enzyme. In this way, water is excluded from the active site and,hence, from the reaction. b: If xylose is used instead of glucose, one water molecule cansqueeze along with it into the active site and then react with ATP, leading to hydrolysis.
instead of glucose, we can fool it into hydrolyzing ATP rather than phospho-rylating the sugar. Xylose looks exactly like glucose with respect to the ring(C1 – C5), so it is able to bind to the active site of hexokinase and induce theactivating conformational change. However, because it lacks the C6 atom withits attached hydroxyl group, it leaves space within the active site for one watermolecule (Figure 3.4b), and it is this water molecule that will react with theactivated ATP in this case.
3.4.2 Phosphohexose isomerase
The mechanism of phosphohexose isomerase is a good example of acid-basecatalysis. The active site contains two basic groups, one of which is protonatedat the start of the reaction (Figure 3.5). Several successive protonations anddeprotonations lead first to ring opening, then to shifting of the double bondfrom the aldo- to the keto-form, and finally to ring closure within the fructose-6-phosphate.
3.4.3 Glyceraldehyde-3-phosphate dehydrogenase
Glyceraldehyde-3-phosphate dehydrogenase provides a straightforward exam-ple of another type of enzyme catalysis, known as covalent catalysis. Thisreaction mechanism is very common in dehydrogenation reactions, for exam-
32 3 Glycolysis
CH
O
CH
CH
CH
O
CH
OH
OH
OH
CH2O-Pho.
H
B+
H
B-
2
CH
OH
CH
CH
O
CH
OH
OH
OH
CH2O-Pho
H
B:
-B
3
H+
CH
OH
CH
CH
O
CH
OH
O
OH
CH2O-Pho
H
B:
B
H
4
H+CH
O
CH2
CH
OH
CH
OH
O
CH2O-Pho
OH
H
:B
B
5
HCH
O
CH2
CH
OH
CH
OH
OH
CH2O-Pho
OH
B+
H
B-
1
CH
O
CH
CH
CH
OH
CH
OH
OH
OH
CH2O-Pho. Glucose-6-P
Fructose-6-P
Figure 3.5 The catalytic mechanism of phosphohexose isomerase. 1: The active sitecontains 2 bases in strategic positions. 2: Glucose-6-phosphate bound the active site,and the ‘electron-hopping’ steps that lead to ring opening. 3-5: Subsequent reactionsteps that lead to migration of the keto group and ring closure of fructose-6-phosphate.
ple in the citric acid cycle and in the β-oxidation of fatty acids. The reactiongoes through the following steps (Figure 3.6):
1. The substrate binds to the active site, where NAD+ is already bound.
2. A deprotonated cysteine thiol group (–S–) in the active site performs anucleophilic attack on the aldehyde carbon, which yields a tetrahedralintermediate state in which the enzyme and the substrate are covalentlybound to each other – hence the term “covalent catalysis”.
3. The intermediate transfers 2 electrons and a proton to NAD+, yieldingNADH and a thioester.
4. NADH leaves and is replaced by NAD+.
5. The thioester is cleaved by a phosphate ion, again by nucleophilic attack.
6. The product (1,3-bisphosphoglycerate) leaves, and the enzyme is restoredto its original state.
The redox cosubstrate used by glyceraldehyde-3-phosphate dehydrogenase,nicotinamide adenine dinucleotide (NAD+), is the major acceptor of hydrogen
3.4 Mechanisms of enzyme catalysis in glycolysis 33
OH
O
O P O
O
O
H
R
OH
OH
OOPOO
O
O P O
O
O
R
O
P
OO
OO
a)
2
R
OHNAD+
HB+
S-
1
R
OH
NAD+
HB+
S-
3
NAD+
HB+
SC
R
O
H
b)
4NADH
HB+
S C
R
O
NAD+
PO
O
OH
OR
O
5
PO
O
OH
O
NAD+
HB+
SC
R
O
6
NAD+
:B
S—H
Figure 3.6 The catalytic mechanism of glyceraldehyde-3-phosphate dehydrogenase.a: All the action occurs at the substrate’s aldehyde group. For simplicity, the remain-der is therefore abbreviated as R in b. The substrate glyceraldehyde-3-phosphate isshown on the left, the product 1,3-bisphosphoglycerate on the right. b: The reactionmechanism. B represents a basic residue in the active site. See text for details.
abstracted from substrates throughout glycolysis and the citric acid cycle. Itsstructure and its reduction by hydrogen are shown in Figure 3.7. As you cansee, all the action occurs at the nicotinamide moiety – the adenosine part iscompletely out of the picture, as far as the redox chemistry is concerned. Why,then, is it there at all? One answer is that it serves as a “tag”, an identifierthat enables the coenzyme to interact with a defined set of enzymes. There
34 3 Glycolysis
N+
P
O
O O
O
P
O
O O
NO N
N
OOH
C
N
NH2
O
OHOH
C
O
NH2
R
a)
R=H: NAD+
R=phosphate: NADP+
S C
R
O
E
CH
CHN
CH
O
NH2
R
HH
CH
CH
CHN
+CH
O
NH2
R
O
C H
R
SE
b)
Figure 3.7 Reduction of nicotinamide adenine dinucleotide (NAD+) byglyceraldehyde-3- phosphate dehydrogenase. a: Structure of NAD+ and NADP+.The redox-active nicotinamide moiety is at the top. The two molecules differ solely bythe lack or presence of a phosphate group at the lower ribose ring. b: The electronsand the hydrogen are transferred to the C4 of the nicotinamide; the electrons thenredistribute within the ring.
is a second, very similar coenzyme, NADP+, which performs the same redoxchemistry but has a different tag with an extra phosphate group and is used bya different set of enzymes. The reasons for this are discussed in a later chapter(see section 9.3).
It is also interesting to note that, as in many other cosubstrates, the tagconsists of a nucleotide instead of a peptide. This likely hearkens back tothe ancient RNA world, in which all catalytic functions are supposed to havebeen performed by RNA enzymes instead of proteins. These RNA enzymes are,for the most part, now extinct, having been replaced by presumably superiorprotein enzymes. However, cosubstrates can’t evolve as easily as enzymes,since they interact with many different enzymes and are therefore are subjectto many more evolutionary constraints; they thus remain frozen in time, likemolecular fossils.
3.4.4 Pyruvate Kinase
The mechanism of pyruvate kinase is similar to that of hexokinase – except thatthe reaction proceeds the other way, producing ATP rather than consuming it.Yet, both reactions are irreversible. How come?
3.5 Energy-rich functional groups in substrates of glycolysis 35
OOH
CH3
O
OOH
CH2
O
H
NO N
N
OHOH
C
N
NH2
OPO
O
O
P
O
O
O
O
P
O
OO
OH
CH2
O
H+
Figure 3.8 Mechanism of the pyruvate kinase reaction. The enolpyruvate formedintermittently tautomerizes to pyruvate; it is this step that drives the reaction in theindicated direction.
The intermediate product of the pyruvate kinase reaction, after transfer ofthe phosphate group from position 2 to ADP, is enolpyruvate. Getting rid of thephosphate group enables the enol group to rearrange itself into a keto group(Figure 3.8). This second step of the reaction is strongly exergonic, and it thuspulls the overall equilibrium of the reaction over to its side. No such exergonicstep occurs with hexokinase, which explains the difference in ∆G.
3.5 Energy-rich functional groups in substrates of glycol-ysis
You will have heard that the most abundant and important energy-rich metabo-lite in the cell is ATP. Within this molecule, the energy is stored in the energy-rich phosphodiester bonds. Cleavage of these bonds is exergonic, and theenergy released in the cleavage drives the various reactions and processes pow-ered by ATP. Conversely, in creating ATP from ADP, we require energy to forma new phosphodiester bond. We just saw where this energy is derived from inphosphoenolpyruvate, and we thus may say that the enolphosphate group isanother energy-rich group.
The first ATP in glycolysis is formed by cleavage of the carboxyphosphatemixed anhydride in 1,3-bisphosphoglycerate, and so we may infer that suchmixed anhydrides are energy-rich groups as well. Indeed, the same group alsooccurs in acetylphosphate and in succinylphosphate, and both of these arecapable as well to drive the formation of a phosphodiester bond in ATP or GTP.
If we look again at Figure 3.6, we see that the mixed anhydride formed fromionic phosphate and a thioester bond. Since ionic phosphate represents a low-energy form of phosphate, it follows that the energy required for the formationof the mixed anhydride must be derived from the thioester, whose formation
36 3 Glycolysis
in turn is driven by the partial oxidation of carbon 1, which changes from thealdehyde form to the more highly oxidized carboxylate form.
In summary, the following functional groups can provide sufficient energyto drive the synthesis of ATP:
1. thioesters – when cleaved,2. aldehydes – when oxidized to carboxylic acids,3. carboxyphosphates – when cleaved, and4. enolphosphates – when cleaved.
3.6 Function of glycolysis under anaerobic conditions
Two molecules of ATP are expended in the initial phosphorylation steps (1 and3 in Figure 3.2). ATP is gained in steps 7 and 10. Since all of the steps from 6 to10 occur twice per molecule of glucose, the net balance is a gain of two molesof ATP per mole of glucose – a very modest number indeed, considering thatthe overall yield in complete oxidative degradation is around 30 moles of ATP.Still, degradation of glucose to pyruvate is a viable source of energy, and it isthe major one that operates in our tissues while oxygen is in short supply, as isthe case in skeletal muscle during maximal exercise such as a 100 meter dash.
To make anaerobic, that is oxygen-free glycolysis feasible, we have to solveone problem. In the glyceraldehyde-3-phosphate dehydrogenase reaction, a mo-lecule of NAD+ is consumed and converted to NADH. Under aerobic conditions,that is when oxygen is available, NADH is reverted to NAD+ in the respira-tory chain. However, under anaerobic conditions, we need another means toregenerate NAD+.
This problem is overcome by the hydrogenation of pyruvate to lactate bylactate dehydrogenase (Figure 3.9a). Even then, as you know from experience,this maximal level of exertion cannot be kept up for long. We will soon haveto slow down as exhaustion and then pain set in. Exhaustion is due to thedepletion of ATP and of glucose, while pain is due to the accumulation oflactate in the tissues and the blood.3
Some cells in the human body, most notably the erythrocytes (red bloodcells), rely entirely on anaerobic glycolysis for ATP production, since they lackthe ability to oxidize pyruvate. Anaerobic glycolysis is a common pathway ofenergy production not only in animals but also in microbes, for example inbaker’s yeast. These face the same problems as human cells do, that is theyneed to regenerate NAD+ and dispose of the acid. The latter task is even morepressing for them than for our us, since they don’t have a blood circulation to
3Measurement of the blood lactate concentration is performed in sports medicine to gaugethe capacity of a trained athlete to sustain aerobic rather than anaerobic metabolism duringprolonged exertion. The anatomical correlate of endurance is not so much the build-up of muscletissue but the extent of its vascularization, that is the abundance of capillaries in the tissue. Ahigh density of capillaries ensures good oxygen supply.
3.7 Transport and utilization of glucose 37
O
OH
CH3
O NAD+NADH+H+
Pyruvate
CH O
CH3
CO2
CH2 OH
CH3
O
OH
CH3
O
CH OH
OH
CH3
O
NAD+NADH+H+
LactatePyruvatea)
b)
Figure 3.9 Regeneration of NAD+ for glycolysis under anaerobic conditions. a: Thelactate dehydrogenase reaction is utilized by mammalians. b: Ethanolic fermentationoccurs in yeast. Pyruvate generated by glycolysis is decarboxylated to acetaldehyde.Reduction of the latter regenerates NAD+ and yields ethanol, which is less toxic thanboth the aldehyde and the lactic acid.
carry away, dilute and buffer the excess acid. One effective strategy, then, is tochemically degrade it. This is the biological purpose of ethanolic fermentation(Figure 3.9b).
3.7 Transport and utilization of glucose in the liver andin peripheral cells
The liver has a special role in many metabolic processes, and prominently soin glucose metabolism. Recall that all the glucose that is taken up from thesmall intestine must pass through the liver first before it can reach any othertissue. The fraction of glucose retained by the liver is regulated depending onthe metabolic situation:• If blood glucose is high, the liver extracts a relatively large fraction and
uses it for conversion to glycogen or for conversion to triacylglycerol (fat).• If blood glucose is low, the liver will only extract small amounts of glucose;
in fact, release of glucose by the liver will exceed its uptake under thesecircumstances.
This latter behaviour is different for example from the brain, which un-abashedly extracts glucose at both high and low glucose levels. This differencebetween the liver and other tissues is implemented at two stages: (1) The uptakeof glucose into the cells, and (2) the phophorylation of glucose to glucose-6-phosphate.
We have discussed before that glucose passes membranes by way of special-ized transporter proteins. Except in the luminal membranes of the gut and of
38 3 Glycolysis
+
+
Figure 3.10 Facilitated diffusion, a form of protein-mediated membrane transport.Typically, substrate binding and dissociation are fast as compared to the conforma-tional change in the transporter protein that leads to release of the substrate on thefar side of the membrane.
the kidney tubules, these transporter proteins operate not by active transportbut by facilitated diffusion, that is they simply speed up transport of glucosealong its concentration gradient (Figure 3.10). Although such transporters arenot enzymes, there is one similarity: With both enzyme reaction and with fa-cilitated diffusion, substrate binding and dissociation are much faster than theaction of the protein – the enzyme reaction, or in this case the conformationalchange required for translocation. Therefore, like simple enzymes, facilitatedtransport obeys Michaelis-Menten kinetics:
V = Vmax[S]
KM + [S](3.1)
You may recall (or else infer from the above equation) that the affinity of theprotein for the substrate, which is represented by KM , controls the dependencyof the activity of on the substrate concentration. In most tissues, the glucosetransporters have a low KM , which means that each transporter molecule isalways operating at full speed,4 irrespective of the glucose concentration. Incontrast, the transporter subtype found in the liver (GLUT2) has a higher KM ,so that the rate of transport is reduced at low glucose concentration.
A similar difference is observed at the stage of glucose phosphorylation.The liver has a special enzyme called glucokinase, which performs the samereaction as does hexokinase but differs from the latter by a higher KM value.Accordingly, phosphorylation will proceed at reduced rate at a low level ofblood glucose (Figure 3.11), and most of the glucose will be allowed to passthe liver and make its way into the general circulation. Conversely, at high
4This does not mean that the overall extraction of glucose always goes at full speed in alltissues. Regulation of glucose uptake in many tissues is mediated by insulin, which changesthe overall number of available transporters but not the activity of the individual transportermolecule (See section 13.1.2).
3.7 Transport and utilization of glucose 39
Glucose (mM)
Glucokinase(liver)
5 10 15 20 25
Hexokinase (most tissues)
V/Vmax
Figure 3.11 Activities of Hexokinase and of glucokinase as a function of glucose con-centration. The physiological range of the glucose concentration is 5-9 mM. Hexokinasewill always be fully active, whereas the activity of glucokinase varies with the substrateconcentration.
concentration, a higher amount of glucose will be extracted by the liver andfed into synthesis of glycogen or fatty acids. The kinetic properties of bothtransport and phosphorylation therefore contribute to the regulatory functionof the liver in glucose metabolism.
Chapter 4
Catabolism of sugars other than glucose
Starch is the most important carbohydrate in our diet. As we have seen, starchconsists of glucose, which is therefore the most important dietary monosaccha-ride. Other quantitatively important sugars are:
1. Lactose (milk sugar) is a disaccharide of glucose and galactose and isfound in milk (surprise!).
2. Sucrose is a disaccharide of glucose and fructose and is found in manyplants and fruit, most prominently in sugar cane and sugar beet.
3. Fructose, as the free monosaccharide, is also found in significant amountsin the diet, both in fruit and as a sweetener.
4. Sorbitol is a sugar alcohol, that is the carbonyl group found in fructose orglucose is reduced to a –CHOH group. It is used as a sweetener but alsooccurs naturally in the diet.
5. Nucleic acids contain ribose and deoxyribose.
Ribose is part of the hexose monophosphate shunt and will be covered in thecorresponding chapter. Here, we will focus on the first three sugars.
The main motif in the metabolism of these sugars is economy: Instead ofcompletely separate degradative pathways, there are short adapter pathwaysthat funnel them into the main pathway of carbohydrate degradation, that isglycolysis. An overview of these pathways is given in Figure 4.1.
40
4.1 Metabolism of sucrose and fructose 41
Fructose 1-P
Fructose
Sucrose
Glucose
Sorbitol
Glyceraldehyde
UDP-GlucoseUDP-Galactose
Galactose 1-PGalactose
Lactose Glucose 6-P
Glucose 1-P
Glyceraldehyde 3-P/Dihydroxyacetone-P
Pyruvate
Figure 4.1 Metabolism of lactose / galactose, sucrose / fructose, and of sorbitol(overview). Short adapter pathways funnel these substrates into glycolysis.
4.1 Metabolism of sucrose and fructose
Sucrose is produced from sugar cane and sugar beet, where it is found in veryhigh concentrations (15-18%). In our “healthy” western diet, it may amount toas much as 20% of dietary carbohydrate. Sucrose contains glucose and fructosejoined in a β-glycosidic bond (Figure 4.2).
Hydrolytic cleavage of sucrose, like that of of maltose, occurs at the surfaceof the intestinal epithelial cells. The enzyme responsible is β-fructosidase, alsonamed sucrase. Both sugars are then taken up by specific transport: Glucose bythe SGLT1 transporter, and fructose by the GLUT5 transporter, which is namedafter glucose but in fact is more active on fructose than on glucose.
Fructose degradation, sometimes called fructolysis, is carried out in theliver. In the first step, fructose is phosphorylated by fructokinase, which usesATP as a cosubstrate. This yields fructose-1-phosphate. The latter is thencleaved by aldolase B, which is found mainly in the liver, in keeping with theliver’s prominent role in fructose degradation. The products of this reactionare dihydroxyacetone phosphate, which is a metabolite in glycolysis, and glycer-aldehyde. Finally, glyceraldehyde is phosphorylated by glyceraldehyde kinase.This yields glyceraldehyde-3-phosphate, which again is an intermediate of gly-colysis (Figure 4.3).
Glyceraldehyde can alternatively be utilized by conversion to glycerol andthen to glycerol-1-phosphate. The latter is a substrate in the synthesis oftriacylglycerol, that is fat. Fructose and sucrose appear to promote obesity
42 4 Catabolism of sugars other than glucose
O
OH
OH
CH2OH
CH2OH
OH O
OH
OH
CH2OH
CH2OH
O
O
OH
OH
OH
CH2OH
2 2
Figure 4.2 Structures of fructose (left) and sucrose. In the sucrose molecule, thefructose is “standing on its head”, and it is the carbon No. 2 that is joined to thecarbon No. 1 of glucose. Glucose is in its α configuration, whereas fructose is in its βconfiguration.
more strongly than equivalent amounts of starch or glucose; if that is the case,the utilization via glycerol-1-phosphate may be among the reasons.
4.1.1 Genetic defects in fructose assimilation
Fructose intolerance is a hereditary disease that is due to a homozygous defectin the aldolase B gene. In this condition, fructose is still phosphorylated by fruc-tokinase. The resulting fructose-1-phosphate, however, cannot be processedfurther, and therefore the phosphate tied up in it cannot be reclaimed. Sincephosphate is required for the regeneration of ATP from ADP, this means thatATP will be lacking, too, which will sooner or later destroy the cell. The diseaseis characterized by potentially severe liver failure (Figure 4.4).
A situation resembling fructose intolerance was observed when fructose wasused as a substitute for glucose in intravenous nutrition of patients that forsome reason could not be fed orally. Application of large amounts of fructoseled to liver damage. Apparently, in these patients, aldolase B could not keep upwith fructose kinase, leading again to the accumulation of fructose-1-phosphateand depletion of phosphate and ATP. Fructose therefore is no longer used as amajor component in intravenous nutrition.
A defect in the gene encoding fructokinase leads to a condition named fruc-tosemia or fructosuria. As these names suggest, fructose levels are increased inthe blood1 and the urine. In this condition, no phosphate depletion occurs, andthe liver cells do not incur any damage. The condition is therefore not severe.
4.2 Metabolism of lactose and galactose
Lactose is the major carbohydrate contained in milk. It is a disaccharide ofglucose and galactose (Figure 4.5). Like maltose and sucrose, it is cleaved atthe brush border of the small intestine, and the monosaccharide fragments areabsorbed and passed along to the liver. The enzyme that accomplishes thecleavage is lactase or, more precisely, β-galactosidase.
1Haima = Greek for blood; hematology is the medical discipline dealing with diseases of theblood.
4.2 Metabolism of lactose and galactose 43
O
OH
OH
CH2OH
OH
CH2OH P O
O
OH
O
O
OH
OH
CH2OH
OH
CH2
CH2
O
OH
CH2
O P O
O
O
CH OH
H
CH2
O P O
O
O
O
CH OH
H
CH2
OH
O
1
2
3
Glyceraldehyde−3−P Glyceraldehyde
Fat
synthesis
Dihydroxyacetone−P
ATP ADP
Fructose
Fructose−1−P
Figure 4.3 Degradation of fructose (fructolysis). 1: Fructokinase; 2: Aldolase B; 3:Glyceraldehyde kinase.
After arriving in the liver, galactose is utilized by conversion to glucose (Fig-ure 4.6a). It is first phosphorylated by galactokinase. The resulting galactose-1-phosphate undergoes an exchange reaction with UDP-glucose, which is cat-alyzed by galactose-1-phosphate uridyltransferase and releases glucose-1-phos-phate and UDP-galactose. Glucose-1-phosphate can be converted by phospho-glucomutase to glucose-6-phosphate (the first intermediate in glycolysis). UDP-galactose is converted to UDP-glucose by UDP-galactose epimerase. This se-quence of reactions constitutes a little metabolic cycle, in which UDP-glucoseand UDP-galactose fulfill a catalytic role but are not subject to any net turnover,much like the intermediates in the citric acid cycle.
4.2.1 Genetic defects in lactose metabolism
A deficiency of the brush border enzyme lactase gives rise to a condition namedlactose intolerance, found frequently in people of East Asian descent past theirinfant age. If lactose is not cleaved, it cannot be absorbed, so it makes its waydown the drain from the small into the large intestine. Many of the bacteriafound there have the capacity to metabolize lactose, which they will happilyconvert to acids and gas. For example, Escherichia coli performs a mixed acidfermentation. One of the products of this fermentation is formic acid (HCOOH),which is then cleaved by formic acid lyase to H2 and CO2.2 This leads to abdom-inal discomfort and diarrhea. Since the environment in the large intestine lacks
2The cleavage of formic acid serves the same purpose, namely detoxification of excess acidderived from fermentation, as does ethanolic fermentation in yeast.
44 4 Catabolism of sugars other than glucose
ATP
Fructose Fructose-1-P
Dihydroxyacetone-P
Glyceraldehyde
+××××
1,3-Bis-P-glycerate
3-P-glycerate
ADP
Figure 4.4 Fructose intolerance. Due to a deficiency of aldolase B, fructose-1-phosphate piles up inside the liver cells, and phosphate is depleted. ATP regenerationfrom ADP stalls, which causes cell damage.
oxygen, H2 generated in the bacterial fermentation is not oxidized but insteadreleased as such, and in part is exhaled (Figure 4.7). An increase in exhaledhydrogen gas upon ingestion of lactose can be used to diagnose the condition.
Treatment consists in omission of lactose in the diet. Milk can be pre-treatedwith purified bacterial β-galactosidase, rendering it suitable for consumptionby lactose-intolerant individuals. Fermented milk products such as yoghurtand cheese are depleted of lactose by microbial fermentation and therefore donot pose a problem for lactose-intolerant individuals.
Other metabolic defects resemble those occurring in fructose metabolism.Two different enzyme defects are known; somewhat confusingly, they are bothreferred to as galactosemia, which means “galactose in the blood”.
1. A defect of galactokinase. In this case, galactose is simply not metab-olized at all; it builds up in the blood and will mostly be eliminated in theurine. The liver will not be adversely affected. However, there is a complicationelsewhere: Cataract (a cloudiness of the lens of the eye). This is believed tooccur by reduction of galactose to galactitol by aldose reductase (see below).
2. A defect of galactose-1-phosphate uridyltransferase. In this case, the sit-uation resembles that outlined above for fructose intolerance: ATP is depletedin the liver cells, because phosphate is trapped in galactose-1-phosphate, andsevere liver damage results.
4.3 The polyol pathway
Sorbitol is not strictly a sugar, since it lacks a keto or aldehyde group. It isnormally a minor component of dietary carbohydrates, but it is also preparedsynthetically and used as a sweetener. In addition, it is formed in our own
4.3 The polyol pathway 45
O
OH
OH
OH
OH
CH2OH
O O
OH
OH
OH
CH2OH
O
OH
OH
OH
CH2OH
Figure 4.5 Structures of galactose (left) and lactose (Galactosyl-β-1,4-glucose).
metabolism from glucose (Figure 4.8). Degradation occurs via dehydrogenationto fructose.
Aldose reductase can also reduce galactose, giving rise to galactitol. It isbelieved that accumulation of sorbitol in diabetes melllitus and of galactitol ingalactosemia occurs in the lens of the eye and in peripheral nerves, and thatthis contributes to the formation of cataract and to nerve damage in the twodiseases. Therefore, inhibitors of aldose reductase are being used—so far withvery limited clinical success—in the therapy of diabetes.
Formation of fructose from glucose via the polyol pathway occurs in theseminal vesicles (part of the male sexual organs), and fructose is found in thesperm. It serves as a supply of fuel to these cells in their quest of an oocyte;the fructose is not utilized by the other tissues the sperm will get into contactwith.
Now, if that is the case, then inhibitors of aldose reductase should be greatas a pill for males, shouldn’t they? However, I have not seen any studies ontheir effects on male fertility.
46 4 Catabolism of sugars other than glucose
O
OH
OH
OH
OH
CH2OH
ATP
ADP
O
OH
OH
OH
CH2OH
OHPO
O
O
O
O
OH
OH
OH
CH2OH
N
NH
O
O
OHOH
COPO
O
O
P
O
O
O
12
O
O
OH
OH
OH
CH2OH
N
NH
O
O
OHOH
COPO
O
O
P
O
O
O
Glucose 1-P
3
a)
b)
O
O
OH
OH
OH
CH2OH
N
NH
O
O
OHOH
COPO
O
O
P
O
O
O
O
O
OH
OH
OH
CH2OH
N
NH
O
O
OHOH
COPO
O
O
P
O
O
O
enzyme+ NAD+
O
OH
OH
O
CH2OH
ORPO
O
O
probable keto-intermediate
Figure 4.6 Metabolism of galactose. a: Pathway. 1: Galactokinase; 2: Galactose-1-phosphate uridylyltransferase; 3: UDP-galactose epimerase. Not shown: Glucose-1-phosphate can be converted to glucose-6-phosphate by phosphoglucomutase. b:Mechanism of UDP-glucose epimerase. The requirement of this enzyme for NAD+
suggests that the epimerization of the hydroxyl group on C4 proceeds via reversibleabstraction of hydrogen from the substrate, that is via a keto intermediate.
4.3 The polyol pathway 47
Lactose
Galactose+
Glucose
LiverLactose
Galactose+
Glucose
⊗⊗⊗⊗
H2
Lung
CO2
Liver
Small intestine
Large intestine
bacterium
Figure 4.7 Lactose intolerance. Left: Normally, lactose is hydrolyzed by lactase, andthe constituent sugars absorbed in the small intestine. Right: In lactose intolerance,lactase is missing, and lactose goes down the pipe into the large intestine, wherebacteria ferment it to hydrogen gas and other metabolites. Exhaled hydrogen can beused as a diagnostic marker for lactose intolerance.
CH
CH
CH
CH
O
OH
OH
OH
CH OH
CH2
OH
CH2
CH
CH
CH
OH
OH
OH
OH
CH OH
CH2
OH
CH2
CH
CH
OH
O
OH
OH
CH OH
CH2
OH
NADPH+H+
NADP+ NAD+
NADH+H+
Figure 4.8 The polyol pathway. Glucose is reduced to sorbitol by aldose reductase(left); sorbitol is then dehydrogenated and turned into fructose.
Chapter 5
Pyruvate dehydrogenase and the citric
acid cycle
As discussed in the preceding chapters, degradation of one mole glucose topyruvate via anaerobic glycolysis only yields two moles of ATP. A much higheryield can be obtained by subsequent complete, oxidative degradation of pyru-vate to CO2 and H2O, which is accomplished by the sequence of pyruvate de-hydrogenase, the citric acid cycle, and the respiratory chain (Figure 5.1). Thesetransformations all occur in the mitochondria, while glycolysis occurs in thecytosol. Therefore, before pyruvate can be completely degraded, it needs tobe transported from the cytosol to the mitochondrial matrix, across the twomitochondrial membranes. While the outer mitochondrial membrane has non-specific pores that allow free permeation of small metabolites, much like theouter membrane of a gram-negative bacterium, the inner membrane is highlyselective and only allows passage of those metabolites for which specific carriersystems exist. Pyruvate is shuttled into the mitochondrion by a specific carriersystem in exchange for hydroxide (OH – ).
5.1 Pyruvate dehydrogenase
Pyruvate dehdrogenase catalyzes the following overall reaction:
pyruvate+ coenzyme A+NAD+ ---------------------------------------→ acetyl-CoA+NADH+H+ + CO2 (5.1)
48
5.1 Pyruvate dehydrogenase 49
Pyruvate
Acetyl-CoA
CO2
Citrate
Isocitrate
α-Ketoglutarate
Succinyl-CoASuccinate
Fumarate
Malate
Oxaloacetate
CO2
CO2
H2
H2O
O2
ADP + Pi
ATP
Glucose
Pyruvate dehydrogenase
Citric acid cycle
Respiratory chain
Pyruvate
Mitochondrion
Mitochondrial transport
CytosolGlycolysis
Figure 5.1 The place of pyruvate dehydrogenase and the citric acid cycle in thecomplete oxidative degradation of glucose. Glycolysis occurs in the cytosol, whereaspyruvate dehydrogenase, the citric acid cycle, and the respiratory chain are locatedin the mitochondria. A specific transport protein located in the inner mitochondrialmembrane is required to transport pyruvate to the mitochondrial matrix.
This reaction does not occur all at once but instead comprises a sequenceof group transfers and redox steps. We can distinguish five steps altogether,which involve an equal number of coenzymes. The five reaction steps occurat three different active sites, which are located on three different types ofenzyme subunits, which are bundled together into one large functional complex.Pyruvate dehydrogenase therefore is a multi-enzyme complex. In each complex,there are multiple copies of each of the three individual enzymes. The numberof subunits totals sixty in the E.coli version of pyruvate dehydrogenase (Figure5.2) and is even higher in mammalian enzymes. Nevertheless, within this largeassembly, each subunit of any type is within easy reach of ones of the othertwo types. This proximity ensures an easy flow of intermediate substrates fromone active site to the next during the sequential stages of the reaction, whichgreatly increases the overall throughput. High throughput is a key advantageof multi-enzyme complexes in general.
50 5 Pyruvate dehydrogenase and the citric acid cycle
SS
NH
O
E3FAD
E1TPP
E2
a)
b)
Lipoamide
Figure 5.2 Structure of the pyruvate dehydrogenase complex. a: Three-dimensionalmodel of the E.coli enzyme. The three different types of subunits are denoted as E1
(grey), E2 (white), and E3 (black). There are 24 subunits each of E1 and E2, arrangedalong the edges of a cube. The 12 subunits of E3 are placed on the planes of the cube. b:For the mechanism of the reaction, all that matters is that the three different types ofsubunits are within easy reach of each other, so that the substrates can be passed alongfrom one active site to the next. Coenzymes: Thiamine pyrophosphate (TPP) and Flavinadenine dinucleotide (FAD) are bound to the active sites of E1 and E3, respectively.Lipoamide is bound to E2, but its long, flexible arm allows it to interact with the activesites of E1 and E3 as well.
The types of subunits are named according to the specific partial reactionsthey catalyze. The first subunit is called pyruvate dehydrogenase, which nametherefore is ambiguous, denoting both the entire complex and a subunit. Thesecond subunit is called dihydrolipoyl transacetylase, and the third one dihy-drolipoyl dehydrogenase. Instead of these explicit names, we will mostly use acommon shorthand notation and call them E1, E2 and E3 in the following.
Figure 5.3 gives an overview of the sequential steps of the pyruvate dehy-drogenase reaction:
1. Pyruvate is decarboxylated, and the remaining hydroxyethyl group be-
5.1 Pyruvate dehydrogenase 51
3
O
O
CH3
O
CO2E1-TPP-
E1-TPP C OH
CH3
SH
SCH3
O
E2
CoA SH
CoA S
O
CH3
E2
SH
SH
E2
S
S
E3-FAD E3-FADH2
NAD+NADH + H+
1
2
4
5
Figure 5.3 Reaction steps catalyzedby the 3 subunits of pyruvate dehy-drogenase. E1-E3 denote the threedifferent types of subunits; numbers1-5 refer to the steps of the reac-tion (see text). TPP: Thiamine py-rophosphate; FAD: Flavin adenine din-ucleotide; CoA: Coenzyme A. E1 andE3 each catalyze two steps, whereasE2 catalyzes one.
comes covalently bound to the TPP coenzyme at E1.2. The hydroxyethyl group is transferred to lipoamide, and is concomitantly
dehydrogenated to an acetyl group. This also occurs in the active site of E1.3. The acetyl group is transferred from lipoamide to coenzyme A. This
occurs in the active site of E2.4. Lipoamide is reoxidized, and the hydrogen is transferred to flavine ade-
nine dinucleotide (FAD) within the active site of E3.5. The reduced form of FAD—FADH2—is reoxidized, and the hydrogen is
transferred to NAD+. This step is catalyzed by E3, too. NADH+H+ is thenreleased from the enzyme.
As you can see, each step in the pyruvate dehydrogenase reaction involves
52 5 Pyruvate dehydrogenase and the citric acid cycle
at least one coenzyme or cosubstrate.1 Therefore, this enzyme illustrates quitenicely the great importance of coenzymes and cosubstrates in enzyme catalysis.
5.2 Catalytic mechanisms in the pyruvate dehydrogenasereaction
The function of the first coenzyme—thiamine pyrophosphate (Figure 5.4a)—consists in providing a carbanion (Figure 5.4b), by which we mean a negativecharge on a carbon atom. Carbanions are very powerful nucleophiles, andthe TPP carbanion functions as such in the decarboxylation of pyruvate. Itattacks the keto group of pyruvate, which leads to a covalent intermediate fromwhich CO2 is cleaved. This yields a second carbanion, now located within thehydroxyethyl group that is the remainder of the substrate. The new carbanioncleaves the disulfide of lipoamide, upon which the entire substrate is cleavedfrom TPP and carried away by lipoamide (Figure 5.4c).
As pointed out above (see Figure 5.2b), lipoamide is covalently attached toE2 but is able to access the active sites of all three types of subunits. Its nextdestination is E2, where transfer of the acetyl group to coenzyme A occurs.This reaction is straightforward, as it consists simply in the formation of onethioester at the expense of another. It proceeds once more by nucleophilicattack of a thiolate anion on a carbonyl group (Figure 5.4).
After the transacetylation from lipoamide to coenzyme A is completed,the only remaining task is to restore lipoamide to its disulfide form. This isaccomplished by E3, which transfers the hydrogen first to FAD (which is tightlybound to the enzyme) and from there to NAD+. The structure of FAD in itsoxidized and reduced forms is shown in Figure 6.5.
5.3 Regulation of pyruvate dehydrogenase
Pyruvate is used in various pathways:1. It can be turned into acetyl-CoA for complete degradation and for fatty
acid or cholesterol synthesis,2. it can be carboxylated to yield oxaloacetate, to be used either in gluconeo-
genesis or in the citric acid cycle, and3. it can be used for the synthesis of amino acids. For example, a single
transamination reaction turns pyruvate into alanine.It is clear then that the activities of all enzymes that act on pyruvate, includ-
ing pyruvate dehydrogenase, have to be regulated in keeping with the prevailingmetabolic needs. Pyruvate dehydrogenase is subject to two modes of regulation(Figure 5.6):
1The last step provides an example of the distinction between coenzymes and cosubstrates:FAD is a coenzyme, since it is in the same state before and after the reaction. NADH is acosubstrate, since it undergoes a net turnover, just like the substrate does.
5.3 Regulation of pyruvate dehydrogenase 53
N
NCH3 NH2
N+
S
CH3
O P
O
O
O
P
O
OH
O
N
NCH3 N
N+
S
CH3
R
H
H
H
H
O
O
N
NCH3N
N+
S
CH3
R
H
H
H
O
O
H+
N
NCH3NH
N+
C S
CH3
R
H
OH
O
a) b)
SH
S
Lip
C
O
CH3
N+
C S
CH3
R1R2
H+
C
O
CH3
N+
S
CH3
R1R2
HS
S
Lip
H+
SH
S
Lip
C
O
CH3
N+
S
CH3
R1R2
H
C
OO
CH3
O
N+
S
CH3
R1R2
H
C
O
O
C
O
CH3
N
S
CH3
R1R2
H
N+
C S
CH3
R1R2
O
OO
CH3
H+
c)
CO2
Figure 5.4 Catalytic mechanism of E1. a: Structure of thiamine pyrophosphate (TPP);b: Electron pushing scheme to account for the formation of the carbanion (C – , blue); c:Mechanisms of decarboxylation of pyruvate, and of transfer of the hydroxyethyl grouponto lipoamide.
1. Allosteric control (see section 2.5). Pyruvate dehydrogenase is stimulatedby Fructose-1,6-bisphosphate but inhibited by NADH and Acetyl-CoA.
2. Phosphorylation by a special regulatory enzyme, pyruvate dehydrogenasekinase. This enzyme is tightly bound to the pyruvate dehydrogenase complex.Phosphorylation inactivates pyruvate dehydrogenase. The kinase, in turn, isallosterically activated by NADH and Acetyl-CoA but inhibited by ADP, NAD+
and by free coenzyme A.
3. Phosphorylation is reversed, and the activity of pyruvate dehydrogenaserestored by a phosphatase, which is also associated with the pyruvate dehydro-
54 5 Pyruvate dehydrogenase and the citric acid cycle
O
P
O
OO
P
O
OO
NO N
N
OHO
C
N
NH2
P
O
OO
C C C C NH
CH3
CH3 OH
H
O
C C
O
NH
C
C S C
O
CH3
CoA S
C
O
CH3
SH
SH
Lip
CoA S
SH
S
Lip
C
O
CH3
H+
SH
S
Lip
C
O
CH3
CoA S H+a) b)
Figure 5.5 The dihydrolipoyl-transacetylase (E2) component of PDH transfers theacetyl group from dihydrolipoamide to acetyl-CoA. a: Structure of acetyl-CoA. Only asmall part—the sulfur atom at the very tip of the molecule—directly engages in theacetyl group transfer. b: Reaction mechanism. One thiol ester bond gives way toanother by way of nucleophilic attack.
PDH (active)
PDH-P (inactive)
Kinase Phosphatase
Ca++
+PyruvateNAD+
CoA-SH
NADHAcetyl-CoA
-
-
+
+
Fructose-1,6-bis-P
Figure 5.6 Regulation of pyruvate dehydrogenase. Plus signs indicate allosteric acti-vation, minus signs allosteric inhibition. PDH-P: Phosphorylated pyruvate dehydroge-nase.
5.4 The citric acid cycle 55
genase complex.All of the above regulatory effects make good physiological sense. NADH
and acetyl-CoA inhibit PDH, which means that the enzyme will slow down whenits products accumulate. Such feedback inhibition is a straightforward way tolink the activity of a pathway to the metabolic requirements it serves. On theother hand, pyruvate, NAD+ and fructose-1,6-bisphosphate apply feed-forwardactivation – as more substrate arrives, the enzyme should pick up speed.
One more interesting detail is that the PDH phosphatase is activated bycalcium ions. Calcium ions are also involved in triggering the contraction ofmuscle cells; activation of PDH will bolster the replenishment of ATP that isconsumed in the contraction.
5.4 The citric acid cycle
With the conversion of pyruvate to acetyl-CoA, the carbon derived from glucosehas reached a central hub of energy metabolism. Degradation of all foodstuffs—carbohydrates, amino acids, and fat—proceeds through this stage. The nextstep toward complete oxidation is the citric acid cycle, or tricarboxylic acidcycle (TCA). Its basic idea consists in releasing the carbon as CO2, and retainingthe hydrogen for “cold combustion” in the respiratory chain. However, if welook more closely, we see that something is missing from this description. Forthe sake of simplicity, let us get rid of the coenzyme A by converting acetyl-CoAto acetate:2
CH3CO-S-CoA +H2O ---------------------------------------→ CH3COOH+ CoA-SH (5.2)
If we look back at figure Figure 5.1, we see that the TCA produces 4 moleculesof H2 and two molecules of CO2. Now, if we attempt to balance the acetate withthese amounts of CO2 and H2:
CH3COOH ---------------------------------------→ 2 CO2 + 4 H2 (5.3)
we see that we are short 4 hydrogens and 2 oxygens on the left side. However,we can balance the equation if we add two molecules of water:
CH3COOH+ 2 H2O ---------------------------------------→ 2 CO2 + 4 H2 (5.4)
Therefore, half of the hydrogen produced in the TCA is gained by the reductionof water. The water-derived oxygen is used to complete the oxidation of theacetyl carbon. Hydrogen derived both from water and the acetyl group is thenre-oxidized in the respiratory chain to generate ATP.
The energy yield of the TCA itself, in terms of directly generated energy-richphosphoanhydride bonds, is very modest – just one molecule of GTP, which
2The hydrolysis of coenzyme A actually takes place at the stage of citryl-CoA, not acetyl-CoA;however, this makes no difference to the overall balance of the TCA.
56 5 Pyruvate dehydrogenase and the citric acid cycle
Figure 5.7 The citrate syn-thase reaction. Abstrac-tion of a proton by a basicresidue in the active site trig-gers nucleophilic attack byacetyl-CoA on oxaloacetate.The reaction is pulled for-ward by the subsequent hy-drolysis of CoA.
C COOH
O
CH2
HOOC
S
C O
CH2
CoA
BH3H+
H+
S
C O
CH2
CoA
C COOH
OH
CH2
HOOC
BH3H+
SH
CoA
BH3H+ C O
CH2
C COOH
OH
CH2
HOOC
O
OH2
S
C O
CH3
CoA
BH2
C COOH
O
CH2
HOOC
is equivalent to ATP, is generated for each molecule of acetyl-CoA degraded,compared to approximately 15 in the respiratory chain. It is clear thereforethat the TCA’s main contribution to ATP generation is to provide H2 for therespiratory chain.
5.5 Reactions in the citric acid cycle
The individual reactions in the TCA are outlined in Figures 5.7 and 5.8:
1. The acetyl group of acetyl-CoA is added to the carbonyl group of ox-aloacetate (Figure 5.7). Coenzyme A is released in the process. This reactionis carried out by citrate synthase. The key aspect of the mechanism is theabstraction of a proton from the acetyl group, which creates a carbanion that inturn reacts with the carbonyl bond of oxaloacetate to create a hydroxyl group.
2. The newly formed hydroxyl group is shifted to an adjacent carbon (Figure5.8a) to yield isocitrate. The latter reaction is catalyzed by citrate isomeraseand involves the transient abstraction of water across the two carbons involved;the water is then added back in the reverse orientation.
3. Isocitrate is decarboxylated and dehydrogenated by isocitrate dehydroge-nase, which yields α-ketoglutarate. In contrast to the pyruvate dehydrogenasereaction, dehydrogenation precedes decarboxylation. This is shown by the oc-currence of the intermediate depicted in Figure 5.8b, known as oxalosuccinate.
5.5 Reactions in the citric acid cycle 57
a) CH2 COOH
COOH
CH2 COOH
OH
CH2 COOH
COOH
CH COOH
CH2 COOH
CH COOH
CH COOHOH
OH2 OH2
b)
CH2 COOH
CH2
C COOHO
NAD+
NADH+H+
Coenzyme A -SH
CO2 CH2 COOH
CH2
C SO CoA
d)CH2 COOH
CH2
C SO CoA
CH2 COOH
CH2
CO O P
O
O
O
CH2 COOH
CH2
CO O
GDP GTPPhosphate
e)
1 2 3
CH2
CH2
COOH
COOH
CH
CH
COOH
COOH
CH
CH2
COOH
COOH
OHFADFADH2
H2O
C
CH2
COOH
COOH
O
NAD+ NADH+H+
c)
CH2 COOH
CH COOH
C COOHO
CH2 COOH
CH2
C COOHO
CH2 COOH
CH COOH
CH COOHOH
NAD NADH+H+ CO2
Figure 5.8 Further reactions in the citric acid cycle. a: The citrate isomerase reactionproceeds via a dehydrated intermediate (called cis-aconitate). b: Isocitrate dehydro-genase transfers hydrogen to NAD+ to generate oxalosuccinate as an intermediate,which it then decarboxylates. c: The α-ketoglutarate dehydrogenase reaction is anal-ogous to the one catalyzed by pyruvate dehydrogenase. d: The succinate thiokinasereaction utilizes the energy-rich thioester bond of succinyl-CoA to generate GTP. Thereaction proceeds via a succinylphosphate intermediate. e: Succinate dehydrogenase(1), fumarase (2), and malate dehydrogenase (3) regenerate oxaloacetate to close thecycle.
58 5 Pyruvate dehydrogenase and the citric acid cycle
There are two isozymes of isocitrate dehydrogenase; one uses NAD+ and theother NADP+ as the cosubstrate. The role of these two isozymes is consideredin section 6.6.
4. α-Ketoglutarate is converted to succinyl-CoA by α-ketoglutarate dehy-drogenase (Figure 5.8c). This step is completely analogous to the pyruvatedehydrogenase reaction. The analogy is reflected in a high degree of homologybetween the subunits of the two enzymes. If you look closely at the mecha-nism (Figure 5.3, above), you will see that the reactions carried out by the finalsubunit (E3) will be identical in both cases, since E3 interacts with lipoamidewhen only hydrogen is left but the rest of the substrate is gone. Indeed, the twoenzyme complexes share the very same E3 protein; only E1 and E2 are specificfor the respective substrates. The same E3 occurs yet again in an analogousenzyme that participates in the degradation of the branched chain amino acids(section 12.5.2).
5. Succinyl-CoA is converted to succinate by succinate thiokinase, and GDPis concomitantly phosphorylated to GTP. We know from the glyceraldehyde-3-phosphate dehydrogenase reaction (Figure 3.6) that thioester bonds are energy-rich and can drive the phosphorylation of carboxylic acids.3 A carboxylic acidphosphate, succinylphosphate, also occurs as an intermediate in the succinatethiokinase reaction (Figure 5.8d). As with phosphoglycerate kinase, the phos-phate group is then transferred to a nucleotide diphosphate. This nucleotideis GDP instead of ADP; however, the energy content of the phosphoanhydridebonds created in GTP and ATP is virtually the same.
6. Succinate is dehydrogenated across the CH–CH bond by succinate de-hydrogenase to yield fumarate (Figure 5.8e, left). The coenzyme used in thisreaction is flavin adenine dinucleotide (FAD). As a rule of thumb, you can as-sume that FAD is used in the dehydrogenation of CH–CH bonds, whereas eitherNAD+ or NADP+ are used in the dehydrogenation of CH–OH bonds. While allother enzymes in the TCA are in aqueous solution in the mitochondrial matrix,succinate dehydrogenase is bound to the inner surface of the inner mitochon-drial membrane; it is identical with complex II of the respiratory chain (seesection 6.1).
7. Fumarate is hydrated to L-malate by fumarase (Figure 5.8e, middle).8. Malate is dehydrogenated by malate dehydrogenase to yield oxaloacetate
(Figure 5.8e, right).
The malate dehydrogenase reaction regenerates oxaloacetate, and thuscloses the cycle. It is noteworthy that its equilibrium favours malate, so thatthe concentration of oxaloacetate is very low. This has two consequences:
1. The availability of oxaloacetate is a kinetic bottle neck that controls therate of the initial reaction of the TCA, that is the synthesis of citrate.
3With glyceraldehyde-3-phosphate dehydrogenase, the reaction proceeds in the opposite direc-tion in glycolysis, but it is reversible and functions in the opposite reaction in gluconeogenesis.
5.6 Regulation of the citric acid cycle 59
2. The low concentration of oxaloacetate also detracts from the free energythat is released by the citrate synthase reaction. To make that reaction goforward, it is necessary to sacrifice the energy-rich bond of the CoA thioester,which in contrast to the succinate thiokinase reaction is simply hydrolyzed andnot utilized for the generation of a molecule of GTP or ATP.
5.6 Regulation of the citric acid cycle
Acetyl-CoA is not only utilized for complete oxidation but also for biosynthetis,most notably of fatty acids and of cholesterol. Therefore, the activity of the cit-ric acid cycle must be regulated in keeping with the prevailing metabolic needs.Major regulatory factors are: (1) The concentration of oxaloacetate, which limitsthe rate of the citrate synthase reaction. (2) Inhibition of isocitrate dehydroge-nase and of α-ketoglutarate dehydrogenase by NADH, the major direct productof the TCA, and by ATP, the ultimate product of complete oxidation via theTCA and the respiratory chain.
Intriguingly, the inhibition by NADH and ATP applies only to the NAD+-dependent isocitrate dehydrogenase, but not to the NADP+-dependent one,which in cells with high TCA activity such as heart and skeletal muscle cells hasa much higher activity than the former. How, then, is this enzyme regulated?This appears to occur in coordination with the flow through the respiratorychain and the proton-motive at the inner mitochondrial membrane. The mech-anism is quite fascinating and is discussed at the end of the following chapter.
Chapter 6
The respiratory chain
In the respiratory chain, the NADH and FADH2 accumulated in the precedingdegradative pathways, mostly the TCA, is finally disposed of by reacting itwith molecular oxygen. The free energy of this “cold combustion” is usedto generate ATP. While a small amount of ATP (or GTP) is produced in thosepreceding pathways, the contribution of the respiratory chain is the largestby far. This is the reason that only aerobic metabolism allows us to sustainphysical exertion for extended periods of time.
The workings of the respiratory chain are quite different from all otherpathways in human metablism (see Figure 6.1). Those other pathways consist ofa succession of discrete enzymatic reactions. In all the ATP-producing reactionsthat we have seen so far, the energy was always passed from one energy-richbond to the next. In contrast, the respiratory chain combines chemical reactionswith physical forces that are not pinned down to individual molecules, and theenergy is stored and converted in novel ways. The entire process consists ofthe following stages:
1. Abstraction of the hydrogen from NADH and FADH2, and separation ofthe hydrogen into protons and electrons.
2. Passage of the electrons down the electron transport chain, which is acascade of redox cofactors that are bound to four large protein complexes em-bedded in the inner mitochondrial membrane. The protein complexes functionas proton pumps; migration of the electrons along this chain makes them expelprotons from the mitochondrion. For each electron migrating down the chain,multiple protons are pumped out of the mitochondrion.
60
6.1 ATP synthesis can be separated from electron transport 61
Q
C
e-H+ H+ H+
H+
O2
H+
H+
ADP + P
ATP
e-
H2O
H+
I
II III IV
H+ H+ H+H+H+ H+
FADH2
e-
NADH
H+
ATP synthase
Mitochondrial matrix
Cytosol
Figure 6.1 Overview of the respiratory chain. Hydrogen provided by NAD+ or FAD issplit into protons and electrons. The electrons are passed along a cascade of proteinslocated in the inner mitochondrial membrane, complexes I–IV, that use the energyprovided by the electron flow to pump protons out of the mitochondrial matrix, intothe cytosol. The electrons then react with oxygen and are rejoined by protons to yieldwater. The protons accumulated outside the mitochondrion flow back in through ATPsynthase, which is thereby induced to rotate. The rotary motion is then harnessed todrive the synthesis of ATP from ADP and ionic phosphate. Q: Ubiquinone (coenzymeQ); C: Cytochrome C. Note that the reactions as shown here are not stoichiometricallybalanced.
3. At the end of the electron transport chain, the electrons are scooped upby oxygen, which then combines with protons to yield water.
4. The protons accumulated outside the mitochondrion are allowed back inthrough another membrane protein, ATP synthase. This protein is a molecularmotor, driven to rotate by the flow of protons into the cytosol. The rotarymotion of ATP synthase in turn drives the synthesis of ATP from ADP andphosphate.
As mentioned before, the outer mitochondrial membrane is permeable formost small molecules and ions, and therefore the proton concentration betweenthe two mitochondrial membranes is in equilibrium with the cytosol. Theproton concentration gradient therefore exists across the inner mitochondrialmembrane only.
If you think that all this sounds rather strange and vague, you are right –but don’t let that trouble you. The purpose here is only to divide and conquer,that is to break up the overall process into manageable parts, which we canthen tackle in more detail in their turn.
62 6 The respiratory chain
a)
b)
Q
C
e- e-
H+ H+
H+ H+
I
II III IV
H+
H+
O
NO2
NO2
O
NO2
NO2
H+
OH
NO2
NO2
OH OH
NO2
NO2
OH
H+ H+
H+
H+ H+
H+
H+
Figure 6.2 Uncoupling of the respiratory chain. a: The decoupling compound dini-trophenol can diffuse across the inner mitochondrial membrane in both its protonatedand unprotonated form. It can therefore carry protons into the mitochondrion, therebydissipating the driving force for the ATP synthase. In the presence of dinitrophenol,electron transport and hydrogen oxidation (molecular respiration) will continue, butATP synthesis will cease. b: The same thing happens with uncoupling proteins.
6.1 ATP synthesis can be separated from electron trans-port
The first thing to note about electron transport and ATP synthesis is that theycan be experimentally separated from each other. Electron transport occurswithout ATP synthesis in the presence of uncoupling agents. These are proton-carrying compounds such as dinitrophenol, which facilitate the transport ofprotons downhill their concentration gradient, that is back into the mitochon-drion.1 This will short-circuit the proton gradient and prevent the ATP synthase
1In general, charged compounds do not cross membranes efficiently. However, dinitrophenolcan cross the mitochondrial membrane not only in protonated, neutral but also in the deproto-nated, charged form. This is because the charge in dinitrophenol is highly delocalized, that isspread across the enrtire molecule.
6.1 ATP synthesis can be separated from electron transport 63
Light
H+
H+H+
ADP+Pi
ATPH+
H+
H+
H+
H+H+
H+
Figure 6.3 The Racker experiment. Bacteriorhodopsin is a light-driven proton pump.A vesicle containing both bacteriorhodopsin and ATP synthase will accumulate protons,which will drive ATP synthase. Note that the orientation of both proteins is inside outrelative to that found in the natural (mitochondrial or bacterial) membranes.
from functioning (Figure 6.2a). The same functional effect is brought about byuncoupling proteins (Figure 6.2b). These are passive proton transporters, lo-cated in the inner mitochondrial membrane and found in particularly highconcentration in a special tissue called brown fat. Their physiological signifi-cance is discussed in section 10.3.1.
The effect of uncouplers shows that electron transport can occur in theabsence of ATP synthesis. On the other hand, ATP synthesis will occur in theabsence of electron transport if another means is provided to sustain a protongradient. An experimental system, devised by Ephraim Racker, is shown inFigure 6.3. Here, a molecule of ATP synthase has been incorporated insideout into a liposome, that is a small artificial membrane particle, along with amolecule of bacteriorhodopsin. The latter is a quite remarkable protein, foundin a certain bacterium (Halobacterium halobium), that functions as a light-driven proton pump. Accumulation of protons inside the liposome will set theATP synthase in motion and drive the synthesis of ATP.
The significance of these findings is that, despite their presence in the samemembrane and their close proximity, the proton gradient is the only functionallink between the electron transport chain and ATP synthase: The electrontransport chain generates the proton gradient, whereas ATP synthase puts it towork and thereby dissipates it. Because of this clear separation, we can safelyexamine these two functions separately from each other.
64
6T
he
respir
ato
ry
ch
ain
TM domain
complex I
complex II
complex III
complex IV
mitochondrial matrix
cytoplasmic side
Figure 6.4 Structures of the four respiratory chain complexes that form the respiratory chain. The structure of Complex Iis only known in part, and a lame outline has been substituted for the missing trans-membrane domain. Redox cofactors arehighlighted. Yellow/grey blobs represent iron sulfur clusters. Organic rings (green) with grey balls (iron atoms) in the centerare hemes; other rings are flavins or ubiquinone. Drawn with pymol from pdb files 3ias, 2fbw, 3cx5 and 2zxw, after a figure inBiochemistry 42:2266–2274 (2003).
6.2 The electron transport chain 65
6.2 The electron transport chain
Figure 6.4 shows structural sketches of the four protein complexes that formthe respiratory chain. Each of the four complexes has a specific role in theelectron transport process:
1. Complex I accepts hydrogen from NADH + H+ and is therefore also calledNADH dehydrogenase. The NADH is oxidized back to NAD+ and thereby read-ied for the next round of reduction in the TCA or by pyruvate dehydrogenase.The two hydrogen protons are expelled, as are apparently two additional pro-tons. The electrons are thereafter transferred to the small carrier moleculeubiquinone (see Figure 6.5c).
2. Complex II accepts hydrogen from succinate. It was mentioned before(section 5.5) that it is identical with succinate dehydrogenase, which illustratesthat the two pathways are really functionally one. The electrons are againtransferred to coenzyme Q, but no proton extrusion occurs at complex II.
3. Complex III reoxidizes coenzyme Q and expels protons. According tothe coenzyme Q cycle model presented below, four protons are being expelledat this stage for each pair of electrons transported, but in some sources thenumber of protons are expelled is given as two. This example shows that thereis still some uncertainty about the mechanistic details of the electron transportchain. The electrons are being delivered to the small electron carrier proteincytochrome C.
4. Complex IV reoxides cytochrome C and is therefore also called cytochromeC oxidase. The electrons are transferred to oxygen, and the considerable freeenergy associated with this electron transfer step is utilized to expel up to 4protons from the mitochondrial matrix.
6.2.1 Redox cofactors in the electron transport chain
Electrons do not occur in free form but are always part of molecules or ions2
Therefore, to make electrons flow along the prescribed path along complexesI-IV, these proteins must provide functional groups that are able of acceptingand donating electrons. These groups must be closely spaced, within a fewAngstroms of each other, to allow for efficient electron transfer. Furthermore,to persuade the electrons to flow in the right direction, the successive transi-tions must be exergonic, that is their free energy (∆G) must be negative.
In Figure 6.4, you can see a multitude of redox cofactors, neatly spaced alongthe protein molecules, that function as “stepping stones” for the migratingelectrons. These prosthetic groups fall into five classes:
2Electrons do occur free as β-particles in ionizing β-radiation. However, to escape capture,β-particles must possess an amount of energy much higher than those available in biochemicalor other chemical reactions. This high energy causes them to break up any molecules in theirpath into ions or radicals.
66 6 The respiratory chain
CH3O
CH3O
O
O
CH3
CH3
H
n
N N
N N
R
R R
R
R
RR
R
a)
b)
d)
Cys Cys
CysCys
Cys
Cys
Cys
Cys Cys
Cys
Cys
Cys
N
N
NH
NCH3
CH3
OH
OH
OH
OH
O
O
P
O
O
O
R
NH
N
NH
NH
CH3
CH3
O
R = H: FMNR = AMP: FAD
c)
Figure 6.5 Structural families of redox cofactors in the respiratory chain. a: Flavinenucleotides (FAD and FMN), oxidized (left) and reduced (right). b: A heme cofactor withan iron atom coordinated by the four nitrogen atoms of the ring. The nature of the ‘R’residues differs in the various heme cofactors, which will contribute to their distinctredox potentials. c: Ubiquinone. The isoprenyl tail is very long (n=10) and hydrophobic,so that ubiquinone is confined to the membrane interior. See Figure 6.8 for details onthe redox chemistry. d: Iron sulfur clusters. 1, 2 or 4 of the sulfurs coordinated toeach iron atom can be part of cysteine side chains; the others will be free sulfide.
6.2 The electron transport chain 67
1. Flavins (Figure 6.5a). We have already encountered flavin adenine dinu-cleotide (FAD) as the redox coenzyme used by succinate dehydrogenase. BothFAD and the related flavin mononucleotide (FMN) occur in the respiratory chain.
2. Porphyrins or hemes (Figure 6.5b). These are tetrapyrrol rings that holda central iron ion which can adopt different oxidation states (mostly Fe2+ andFe3+, although Fe4+ occurs within complex IV). The name “cytochrome” is insome cases applied to the hemes themselves, in others to the entire complexof the heme and the protein it is bound to.
3. Iron-sulfur clusters. Here, it is again the iron that accepts and donateselectrons by alternating between different oxidation states. Each iron ion is heldin place by four sulfur atoms. These can be cysteine sulfurs or free sulfides(S2-) in various proportions, which gives rise to different sizes of iron-sulfurclusters (Figure 6.5c).
4. Cytochrome C oxidase (complex IV) contains copper ions that function asredox cofactors in the final transfer of electrons to oxygen. These copper ionsare coordinated—that is, bound—directly by amino acid side chains, withoutany additional prosthetic group.
Since there are many more than four individual cofactors in the respira-tory chain, most classes occur in multiple instances. Importantly, the redoxpotentials (see below) of the different cofactors that belong to the same chem-ical class can be different due to influences from their respective molecularenvironments.
In addition to the stationary redox cofactors that occur within complexesI-IV, there are two electron carriers that are not tighly associated with oneindividual complex but function as shuttles between them:
1. Ubiquinone or coenzyme Q (Figure 6.5c). This coenzyme contains aquinone group. It carries electrons, as hydrogen, from complexes I and IIrespectively to complex III. It also contains a long hydrophobic poly-isoprenetail, which confines it to the hydrophobic interior of the membrane. Its specialmode of reoxidation is considered in some detail below (Figure rchainQcycle).
2. Cytochrome C. This is a small protein that again contains a heme. It islocated at the outer surface of the inner mitochondrial membrane and shuttleselectrons between complex III and complex IV.
6.2.2 Electrochemistry of the respiratory chain
It was stated above that the free energy of the transfer of an electron from onecofactor to the next must be negative in order to make it happen. Another wayof saying the same thing is that the cofactors should have progressively higherredox potentials. The reason why redox potentials are commonly used in thiscontext is that they can be measured more directly than ∆G. However, free
68 6 The respiratory chain
12 H2 H+
e–
e–
e–
e–
Q Q+
∆E′0 > 0a)
2 H+ H2
2 e– 2 e–
NADH NAD+
+ H+
e–
e–
e–
∆E′0 < 0b)
Figure 6.6 Experimental setup for measuring standard potentials. The sample andthe reference are contained in two adjacent chambers. Platinum electrodes are im-mersed in the solutions. Electrons are withdrawn from one chamber and delivered intothe other, flowing through a voltmeter indication the direction and magnitude of thepotential difference. Protons and other ions can flow across a salt bridge to maintainelectroneutrality. Left: Coenzyme Q withdraws electrons from the standard hydrogenelectrode and accordingly has a positive ∆E′0. Right: NADH feeds electrons into thestandard electrode, making its ∆E′0 positive.
energy and redox potential are directly related by the following equation:
∆G = −∆E nF (6.1)
where ∆E is the difference in the redox potentials between two cofactors, Fis Faraday’s constant: 96,500 Coulombs/mol, and n is the number of electronstransferred.3 For example, NADH feeds two electrons at a time into the chain,so the value of n for this reaction is 2.
One can look at the redox potential of a cofactor as its affinity for electrons– the higher it is, the more strongly the cofactor will attract electrons.4 Theredox potential is a useful parameter since it can readily be measured. Figure6.6 shows the required experimental setup:
1. A chemically inert electrode, often platinum, is immersed in a solutionthat contains standard amounts of the reduced and oxidized forms of ourcarrier,
3Another thing to remember is that the unit of ∆E, Volt, is defined as J/Coulomb, since voltage= energy/charge.
The minus sign in this equation results from the fact that the electron-donating electrode, thecathode, is considered negative by convention. It is fundamentally meaningless but handy as atrap in exam questions.
Faradays constant can be considered a historic artifact, resulting from the fact that units ofelectricity were already defined before the principles of electrochemistry were understood.
4You have encountered the same concept with chemical elements as their electronegativity:An element with a high electronegativity holds on to electrons particularly firmly, that is it has ahigh affinity for electrons.
6.2 The electron transport chain 69
2. a reference electrode is immersed in a bath of an electron carrier of knownredox potential, and
3. a voltmeter connects the two compartments.
Another connection must be made between the two baths so as to permit flowof ions to preserve electroneutrality but prevent mixing of the contents byconvection. This may be a small hole plugged with agar.
The standard reference electrode commonly used in chemistry is the plat-inum/hydrogen electrode. This electrode contains hydrogen both in reducedform—H2, kept constant by equilibration of the aqueous phase with hydrogengas at a pressure of 1 atmosphere—and in the oxidized form, H+, which isadjusted to 1 mol/l or pH 0. Platinum functions both as a conductor and as acatalyst for the interconversion of reduced and oxidized hydrogen.
The potential of a redox carrier measured against this electrode is definedas its standard redox potential or ∆E0. For biochemical purposes, a modifiedstandard electrode is used, which has a pH of 7 instead of 0, and the redoxpotentials measured against this electrode are referred to as ∆E′0. A pH of 7 isjust as arbitrary a choice as pH 0, but we will stick with it because the booksdo so, too.
The redox potentials of several selected carriers in the respiratory chain areshown in Figure 6.7. The lowest value is found with NAD+, in keeping withits position at the start of the transport chain. The next carrier in sequence,FMN, is part of complex I. It has a slightly higher potential than NADH and istherefore able to abstract its electrons.
The iron-sulfur cluster N2, which occupies the lowermost position withincomplex I as shown in Figure 6.4, has a significantly higher potential than theFMN. This step in potential corresponds to a significant amount of free energythat is released at some point between FMN and N2. Complex I uses this energyto expel protons from the mitochondrion, against their concentration gradient.Major steps in potential that drive proton expulsion also occur within complexIII and complex IV.
Only minor steps of potential occur in all other stages, including the deliveryof electrons from complex I and complex II—the latter is represented in thefigure by FADH2—to complex III via ubiquinone, and between complexes IIIand IV via cytochrome C. These minor steps suffice to drive electron transportforward, but they are too small to drive any additional external work (seebelow).
The redox potential increases continuously along the respiratory chain toreach its highest value at oxygen, which therefore has the highest affinity forthe electrons and gets to keep them. Reduced oxygen, which recombines withprotons to yield water, therefore is the end product of respiration.
While the scale of redox potentials establishes the general direction of elec-tron flow, the redox cofactors also differ in two other important aspects:
70 6 The respiratory chain
−0.4
−0.2
0
0.2
0.4
0.6
0.8
1.0
75.2
36.6
−2.0
−40.6
−79.2
−117.8
−156.4
−195.0
NADH FMN
(Fe-S)N-2CoQ
Heme c1 CytC
Heme a3
O2
FADH2 (complex II)complex I
complex III
complex IV
∆E′ 0(V
)
∆G(K
J /2
mol
elec
tron
s)
Figure 6.7 Redox potentials for selected cofactors along the respiratory chain. Elec-trons are fed either from NADH into complex I or from FADH2 into complex II. Thescale on right hand side shows the conversion of ∆E to the molar ∆G, per pair of elec-trons (see equation 6.1). As ∆E goes up, ∆G goes down, driving the electron transportforward. Major jumps in potential occur within complexes I, III and IV; these are thesites that harness the free energy of electron transport to proton export. Abbreviations:CoQ, coenzyme Q (ubiquinone); CytC, cytochrome C; Fe-S, iron sulfur cluster.
1. NADH, FADH2, FMNH2 and coenzyme Q carry both electrons and protons– that is, hydrogen. In contrast, the hemes and the iron-sulfur clusters carryonly electrons.
2. NAD+ can only accept and donate pairs of electrons, whereas the hemesand iron-sulfur clusters can only accept and donate single electrons.
The switch from the two-electron carrier NADH to the one-electron carryingFe−S clusters within complex I is mediated by FMN, which can accept or donateelectrons both pairwise and singly:
NADH+H+ + FMN ---------------------------------------→ NAD+ + FMNH2 (6.2)
FMNH2 + Fe III−S ---------------------------------------→ FMNH · +H+ + Fe II−S (6.3)
FMNH · + Fe III−S ---------------------------------------→ FMN+H+ + Fe II−S (6.4)
After accepting H2 from NADH + H+ (equation 6.2), FMNH2 donates theelectrons one by one to the first Fe−S cluster (equations 6.3 and 6.4), adoptinga sufficiently stable radical form between these two transfers.
The electron transfer between FMNH2 and Fe−S also illustrates what hap-pens if an electron-only carrier is reduced by a hydrogen carrier: The protonsare simply shed into the solution. Conversely, when a hydrogen carrier is re-duced by an electron carrier, as happens with the reduction of ubiquinone by
6.2 The electron transport chain 71
(Fe–S)N2, protons are taken up from the solution:
Fe II−S+H+ +Q ---------------------------------------→ QH · + Fe III−S (6.5)
Fe II−S+H+ +QH · ---------------------------------------→ QH2 + Fe III−S (6.6)
Hydrogen carriers alternate with electron-only carriers at several points inthe chain. This means that electrons are stripped of their protons and rejoinedby protons again repeatedly during transport. Where protons are strippedoff, they are preferentially released at the cytosolic side, whereas new protonsare taken up from the mitochondrial side. This accounts for some of theproton translocation activity of the respiratory chain. As an example of of theforegoing, we will have a look at the (in)famous ubiquinone cycle.
6.2.3 The ubiquinone cycle
Ubiquinone carries hydrogen from complexes I and II to complex III, whichin turn passes them on to complex IV. Complex III has two binding sites forubiquinone, and both of them are occupied while the ubiquinone cycle runs.The cycle goes through the following steps (Figure 6.8):
1. We start with a reduced ubiquinone (QH2) bound to the first site, and anoxidized ubiquinone (Q) bound to the second site.
2. The protons of QH2 are stripped off and expelled at the cytosolic side.One electron is passed on first to a heme within complex III and then tocytochrome C; the other electron is passed on to the second molecule ofubiquinone.
3. The first ubiquinone, now oxidized to Q, is then replaced by a new QH2
that has been reduced in the preceding steps of the respiratory chain.4. The protons and electrons of the new QH2 are abstracted and split as in
step 2.5. The reduction of the second ubiquinone is now complete. It recombines
with mitochondrial protons to form QH2.6. Q and QH2 switch places and thereby complete the cycle.
Therefore, with each molecule of ubiquinone reduced in the respiratory chain,the two protons it carries are expelled into the cytosol, and two additionalprotons are taken up from the mitochondrial matrix and expelled into thecytosol as well. Complex III uses ubiquinone in a dual role – as a carrier thatconnects it with the preceding part of the respiratory chain, and as a prostheticgroup to facilitate the movement of protons across the membrane.
If you compare the outline of the ubiquinone cycle given here to the descrip-tion in your textbook, you might find the similarity rather remote. In reality, asyou can see in Figure 6.4, complex III contains several more redox co-factorsthat act as intermediate stepping stones in the electron transfer steps outlinedabove. They have been skipped here for simplicity.
72 6 The respiratory chain
CH3O
CH3O
O
O
CH3
CH3
H
n
C CH3O
CH3O
O
O
CH3
CH3 n
H
CH3O
CH3O
OH
OH
CH3
CH3 n
H
CH3O
CH3O
O
O
CH3
CH3 n
H
e-
2H+, e-
a)
b)
QH2
Q
2 H+
Q
Q-
e-QH2
QH2
Q-
2 H+
2 H+
Q
QH2
e-
CytC
CytC
Figure 6.8 The ubiquinone cycle, criminally simplified. a: Redox chemistry ofubiquinone (coenzyme Q). It can accept two electrons successively and two protonssuccessively. b: The ubiquinone cycle (simplified so that I can understand it). ComplexIII has two binding sites for ubiquinone . Oxidation of one molecule of ubiquinonepartially reduces the second one; oxidation of a new molecule of ubiquinone com-pletes reduction of the second one and causes uptake of two protons from the cytosol.Protons released by oxidation are at all times released at the cytosolic side.
6.2 The electron transport chain 73
6.2.4 Cytochrome C oxidase (complex IV)
Cytochrome C is a small hemoprotein that shuttles electrons from complexIII to complex IV. The latter complex, which is also known as cytochrome Coxidase, completes the transfer of electrons by delivering them to oxygen. Inthe process, it pumps some more protons out of the mitochondrial matrix. Thereduction of oxygen is the trickiest part of the entire respiratory chain, as ittakes a full 4 electrons to reduce one molecule of oxygen (O2) to water. Sincethe electrons only arrive one at a time by way of cytochrome C, the reductionwill involve partially reduced oxygen species. These are highly toxic when letloose upon the cell, so the enzyme must provide for stable accommodation forall intermediate stages in the reduction.5
Cytochrome C oxidase solves this problem by clamping the oxygen betweena heme- bound iron and a histidine-coordinated copper, both of which functionas redox cofactors in the reduction of oxygen. The intermediate stages ofreduction are outlined in Figure 6.9.
6.2.5 How is electron transport coupled to proton pumping?
As pointed out above, some of the protons that are being expelled from themitochondrion are accepted from the hydrogen carriers NADH and ubiquinoneand travel together with electrons for a part of the journey. However, at somepoint they must part company, and the protons must be expelled, whereasthe electrons are retained. Also, more protons are being expelled than can beaccounted for by the hydrogen carriers. In particular, complex IV does notinteract with any hydrogen carriers yet expels up to four protons for each pairof electrons accepted. So, there must be mechanisms that extract energy fromthe transfer of proton-less electrons and apply it towards the expulsion ofelectron-less protons. How does this work?
The experimental evidence on this point is pretty fragmentary, and to theextent they are understood the emerging mechanisms are quite complex. There-fore, instead of trying to describe them faithfully, I will present a simplifiedconceptual model to provide an idea of how things work (Figure 6.10).
The basic idea is that binding and release of electrons cause conformationalchanges to a protein. This is entirely analogous to conformational changescaused by allosteric effectors binding to enzymes, or to phosphate groupsbound to proteins (e.g. myosin light chain). An electron carries a charge, acharge causes a field, and a field will cause forces that act on charged residueson the protein, so it is quite easy to see how migrating electrons can causeconformational changes.
5The toxicity of partially reduced oxygen species such as O –2 is actually exploited by phago-
cytes, which release them intra- and extracellularly to destroy microbes. Enzyme defects in theproduction of these reactive oxygen species produce severe immune deficiencies.
74 6 The respiratory chain
N N
N N
R
R R
R
R
RR
R
HisHis
His
O
O
a)
b)
2+
1+
3+
2+
O-
O-
e-
H+
4+
2+
O2-
O2-H+
3+
2+
O2-
O2- H+
H+
e-
H+
2+
1+
2 H+
2 H2O
3+
2+
2 e-
O2
O
O
iron
copper
iron
copper
Figure 6.9 Reduction of oxygen by cytochrome C oxidase. a: Structural features ofthe active site. Iron is part of heme a3, copper is coordinated by three histidine residues.Oxygen is bound longitudinally between them. b: Stages of oxygen reduction. BoundO2 is reduced to peroxide at the expense of both iron and copper. Uptake of an electronand abstraction of a further one from iron reduces both oxygen atoms to the final level(2–). Uptake of protons generates first hydroxyls and then water, and uptake of furtherelectrons restores iron and copper to their original oxidation levels.
6.2 The electron transport chain 75
+
–
+
–
+
mitochondrial matrix
cytosol+
–
+
+ +
Figure 6.10 A conceptual model linking electron transport to proton pumping. Theproton pump sits between two other elements of the electron transport chain. Itpossesses three electron carrier cofactors (white circles). In the resting state, theproton binding site is open to the mitochondrial matrix. Arrival of an electron at thefirst carrier causes a conformational change that everts the proton binding site, so thatit now communicates with the cytosol and ejects the proton, and at the same timefacilitates migration of the electron to the next cofactor. The pump then reverts to itsresting position.
An obvious limitation of the model presented in Figure 6.10 is that it linksthe transport of a single proton to the transport of a single electron. If thenumber of protons is higher, we can account for it by postulating multiplesimilar valves connected in series witin a single respiratory chain complex.
6.2.6 Stoichiometry of proton ejection
It is commonly stated that about 10 protons are ejected for each pair of elec-trons abstracted from NADH, such that 4 protons are ejected at each of com-plexes I and IV, and 2 at complex III.6 Complex II does not eject any protonsbut just abstracts them from FADH2 and passes them on to ubiquinone. If youlook at Figure 6.7, you will notice that the difference in the redox potentials ofFAD and ubiquinone is rather small. Consequently, the amount of free energyassociated with the transfer of electrons from FAD to ubiquinone is too smallto permit the performance of work against the proton gradient.
6Note that this number is at variance with the model of the coenzyme Q cycle given above.Generally speaking, the figures given for the numbers of protons ejected at each stage vary quitea bit between various texts.
76 6 The respiratory chain
6.3 ATP synthesis
Most of the ATP that results from complete oxidative degradation of glucose issynthesized only after the substrate has already vanished in the form of CO2
and H2O. At this stage, the entire available energy is stored in the so-calledproton-motive force across the inner mitochondrial membrane. ATP synthesisis powered by the protons that yield to this force and are pulled back into themitochondrion.
6.3.1 The proton-motive force
The proton concentration in the cytosol is approximately ten times higher thanthat in the mitochondrial matrix. How much free energy does it generate ifprotons travel downhill this concentration gradient? This can be determinedfrom the following formula:
∆G = RT ln [H+]in[H+]out
(6.7)
With R = 8.31 J/K mol, T = 310K, and [H+in]/[H+out] = 10 this comes to roughly6 kJ/mol. While this is significant, the larger contribution to the proton-motiveforce comes from the electrostatic membrane potential across the inner mito-chondrial membrane. Like the proton concentration gradient, this electricalpotential is a direct consequence of the proton pumping: Each proton ejectedleaves a deficit of a positive charge, or one negative charge, inside the mito-chondrion. In a fully energized mitochondrion, the resulting potential amountsto 150 mV, negative inside. The free energy that this membrane potential con-fers to each proton can again be calculated from equation 6.1 and works out toapproximately 15 kJ/mol. In summary, the proton-motive force is caused roughlyto 3/4 by the membrane potential, and to 1/4 by the proton concentration gradi-ent.
6.3.2 ATP synthase
This complex and fascinating molecule is both an enzyme and a molecularmotor.7 It works as follows (Figure 6.11):
1. Protons flow between the a-subunit and the F0-subunit of the ATP syn-thase, which causes the F0 subunit and the γ-subunit attached to it to rotateversus the rest of the molecule (Figure 6.11a).
2. The γ-subunit rubs against the inner circumference of the αβ-hexamer.Because of the asymmetric shape of the γ-subunit, this causes the α- andβ-subunits to undergo cyclical conformational changes (Figure 6.11b).
7A similar molecular motor drives the rotation of the flagellae found in many bacteria, whichenables them to swim. Remember that mitochondria are of bacterial origin.
6.3 ATP synthesis 77
γ
α
β
β
β
α α
ATP
P
AD
P
ATP
a)
b)
c)
F0
γ
α β
δ
a
H+
Figure 6.11 Structure and func-tion of ATP synthase. a: Side view.The F0 and the wurst subunits arerotationally mobile against therest of the molecule. The protonflow occurs at the interface of thea and F0 subunits. b: Top view.The γ-subunit rubs against the α-and β-subunits and subjects themto cyclical changes of conforma-tion. c: Coupling of rotation toATP synthesis. One of the con-formations of β accepts ADP andphosphate, which spontaneouslyreact to form ATP; this is drivenby the unusually high affinity ofthe next conformational state forATP. The rotation of γ forces thetransition to the next conforma-tion, which expels ATP and makesβ available for the next turnover.
3. The β-subunit, which contains the active site of the enzyme, utilizes theenergy transmitted by these conformational changes to synthesize ATP (Figure6.11c).
How does the β-subunit transform the energy of rotation into chemicalenergy for ATP synthesis? Strictly speaking, it doesn’t – it turns out that theisolated β-subunit can create ATP all by itself. In doing so, the β-subunit adoptsdistinct conformational states. In the first state, it binds ADP and phosphate.Once both are bound, β transitions to the next state, which binds ATP withexceptionally high affinity but no longer binds ADP and phosphate. Formationof the several non-covalent bonds between β and ATP provides the energy that
78 6 The respiratory chain
Figure 6.12 A hypothetical model for coupling of proton flux and ATP synthaserotation. See text for details. Source: P. Boyer, Nature 402:247–249 (1999).
is needed to form the single new phosphodiester bond in ATP.
Now with the isolated β-subunit, we have reached a dead end – ATP isbound so avidly as to never be released, so that no further turnover can occur.It is at this stage that the γ-subunit comes into play. Rotation of γ forcesanother change of conformation upon β that in turn kicks out the ATP of theactive site (Figure 6.11c). The force applied by γ on β must be so strong as toovercome and offset the large binding energy that ties the ATP to the enzyme.In summary, the formation of ATP proceeds spontaneously inside β, and theenergy of rotation is applied to force out the avidly bound ATP and reset β forthe next round of catalysis.
The last remaining puzzle is how the proton flux actually promotes rotationof the F0- and γ-subunits. A hypothetical model is illustrated in Figure 6.12. TheF0-subunit consists of 10 c-chains arranged in a pie-slice fashion. Each of themhas a strategic aspartate residue that faces the surrounding lipid membrane.When a given c-chain encounters the a-subunit, the aspartate first connectsto a proton-conduction channel (across a) that allows it to discharge a proton,which it had picked up during the last rotation, and then to another channelthat causes it to accept a new proton from the cytosol.
While there is indeed evidence for alternating accessibility of several strate-gic amino acid residues from the two opposite sides of the membrane, thismodel has a problem: While it tells us how rotation of ATP synthase coulddissipate the proton gradient, it does not tell us how ATP synthase actuallyderives any torque from this, so that it can perform work against resistance.More specifically, why would the rotor always keep going in the same direction
6.4 The ATP yield of oxidative glucose degradation 79
instead of just oscillating? This would save it the trouble of working whilestill permitting proton flux. A steam engine gets around a similar problem byinertia, which is created by adding a nice, heavy, cast-iron flywheel. That’s notpossible here, because of the minuscule dimensions.8 Accordingly, there mustbe a tighter coupling of proton flux and rotation that most likely involves someconformational flexibility in both the a and the c subunits. There are someexperimental data to support this assumption.
There is, however, one interesting finding that the model in Figure 6.12 doesaccount for: The number of protons transported per rotation is identical tothat of the c subunits in F0. Intriguingly, ATP synthases in different organismsvary in their number of c subunits. This will directly affect the stoichiometry ofATP synthesis and proton transport. It would be interesting to know whetherthere are complementary variations in the number of protons driven out perelectron during electron transport. If not, the different subunit stoichiometryof F0 should directly translate into a different ATP yield in the entire respiratorychain.9
6.4 The ATP yield of oxidative glucose degradation
We can now determine how much ATP is produced through the complete oxi-dation of glucose via glycolysis, TCA, and respiratory chain. Here are the bitsand pieces:
1. For each molecule of glucose, a total of 10 moles of NADH and 2 FADH2
are produced by glycolysis, pyruvate dehydrogenase, and citric acid cycle.
2. 10 protons are exported per NADH, and 6 per FADH2 in the respiratorychain, for a total of 112 protons.
3. Each revolution of ATP synthase consumes 10 protons and generates 3ATP.
Overall, we obtain 112 protons per glucose × 3 ATP per 10 protons, or33.6 ATP per molecule of glucose. Therefore, 33.6 ATP could be generatedin the respiratory chain for each molecule of glucose degraded, if all protonswere available for driving ATP synthase. However, some protons are divertedto other purposes, so that the actual yield will be lower than this theoreticalvalue. Most importantly, some protons are needed for ATP transport. ATPsynthesized in the mitochondrion needs to be exported to the cytosol, andADP produced there needs to get back in. This is accomplished by a special
8A student who took this class in 2005, Kelvin Cheung, took up the challenge to calculate thekinetic energy of rotating ATP synthase; it works out to about one billionth of the energy requiredfor making 1 ATP. Honorable mention.
9If you are interested in this problem, you are welcome to dig up and some recent informationon this and discuss it with me. If it merits inclusion here, it will earn you an honorable mentionand a recommendation letter.
80 6 The respiratory chain
transporter protein in the inner mitochondrial membrane that exchanges ATPand ADP for each other.
Since ATP carries one more negative charge than ADP (ATP4– vs. ADP3–), thisexchange amounts to a net export of one negative charge, or to the net importof one positive charge per ATP. The total need of protons per ATP synthesisplus export to the cytosol is therefore approximately 4,10 so that the actualATP/glucose ratio is closer to 28 than to 33. Together with the 4 molecules ofATP and GTP generated in glycolysis and the TCA, the overall yield of ATP permolecule of glucose is approximately 32.
It is worth noting that the extra proton expended on the export of ATP isnot ‘wasted’, as some textbooks lament, but can be considered well spent. Itenables the transport of ATP and ADP against their concentration gradients,allowing for the maintenance of a high ATP/ADP ratio in the cytosol, which willhelp all ATP-consuming reactions there to go at speed, and a higher ADP/ATPratio in the mitochondrion, which will help the ATP synthase to go at speed. Itis kind of like driving on the highway: Sure, you could save fuel by driving at60 km/h all the time, but would you?
6.5 Auxiliary shuttle systems for the re-oxidation of cy-tosolic NADH
In chapter 3, it was mentioned that under aerobic conditions the NAD+ con-verted to NADH by glyceraldehyde-3-phosphate dehydrogenase is re-oxidizedin the respiratory chain. However, NADH cannot pass the inner mitochondrialmembrane, and in fact not even the more porous outer membrane. So, howthen is its oxidation accomplished?
It turns out that NADH is not translocated at all but is re-oxidized, or dehy-drogenated, in the cytosol. The hydrogen is then brought to the mitochondrionby other carriers. This is accomplished by several shuttle systems, in a some-what roundabout manner. The shuttles tie together several enzyme activitieswith specific transporters in the inner mitochondrial membrane (Figure 6.13).
One enzyme that regenerates cytosolic NAD+ is cytosolic malate dehydroge-nase, which reduces oxaloacetate to malate. Cytosolic malate is then exchangedfor mitochondrial α-ketoglutarate by a specific transporter and dehydrogenatedback to oxaloacetate inside the mitochondrion.
The question whether or not oxaloacetate can leave the mitochondrion inexchange for α-ketoglutarate is, in my opinion, not settled. If it can, it ispossible to draw a pretty simple shuttle mechanism, shown in Figure 6.13a. Ifit cannot, we have to use transamination (see section 12.2) as a workaround:
10It is not actually a proton; therefore, in my understanding, it is only the membane potentialcomponent but not the proton concentration component of the proton- motive force that getsdissipated. Then, it costs about 3/4 of the energy of a pumped proton to export one ATP.
6.5 Shuttle systems for the NADH re-oxidation 81
AspartateAspartate
Glutamate Glutamate
α-Ketoglutarate α-Ketoglutarate
Oxaloacetate Oxaloacetate
Malate MalateNADH+H+
NAD+ NAD+
NADH+H+
b)
α-Ketoglutarate α-Ketoglutarate
Malate Malate
Oxaloacetate
NADH+H+
NAD+
Oxaloacetate
NADH+H+
NAD+
a)
Q
I
II
III
IV
C
2 e-
DHAP
Glycerol-P Glycerol-P
DHAP
NADH+H+
NAD+
2 H+
GPD(FAD)
c)
Figure 6.13 Shuttlesystems for the transferof NADH equivalentsfrom the cytosol to themitochondrion. a: Afairly simple, hypotheticalshuttle that is not in thetextbooks. b: The malate-aspartate shuttle. c: Theglycerophoshate shuttle.Abbreviations: DHAP, dihy-droxyacetone phosphate;GPD, glycerolphosphatedehydrogenase. Continu-ous gray bars representthe inner mitochondrialmembrane; the broken barin c represents the outermitochondrial membrane.See text for further details.
Oxaloacetate is transaminated using mitochondrial glutamate and the resultingaspartate exchanged for cytosolic glutamate. In the cytosol, transamination isreversed, which closes the cycle. This textbook-approved cycle is known as themalate-aspartate shuttle (Figure 6.13b).
In the glycerolphosphate shuttle, the hydrogen is never actually transportedto the mitochondrion. Dihydroxyacetonephosphate serves as the intermedi-ate hydrogen acceptor and is reduced in the cytosol to glycerolphosphate byglycerolphosphate dehydrogenase. Glycerolphosphate traverses the outer mi-tochondrial membrane and reaches the surface of the inner one, where it is
82 6 The respiratory chain
converted back to dihydroxyacetonephosphate by a second dehydrogenase,which abstracts the electrons and feeds them into the respiratory chain at thelevel of ubiquinone (Figure 6.13c). This is similar to the activity of succinatedehydrogenase, and as with the latter, FAD is the coenzyme employed by themitochondrial glycerolphosphate dehydrogenase.
The glycerolphosphate shuttle bypasses complex I in the respiratory chainand therefore induces ejection of four fewer protons from the cytosol. How-ever, this shortfall is partially compensated for by the two protons that staybehind in the cytosol (or more accurately, the intermembrane space) when theelectrons get abstracted from glycerolphosphate. While this shuttle is some-what less energy-efficient than the malate-aspartate shuttle, it certainly is morestraightforward than the latter, since it avoids all substrate transport across theinner mitochondrial membrane. Remember that inside the mitochondrion thefree concentration of oxaloacetate is low; thus, oxaloacetate probably formsthe bottleneck in the malate-aspartate shuttle. It is interesting to note thatthe glycerolphosphate shuttle is highly active in insect muscle, which has anextremely high ATP turnover during flight.
6.6 Regulation of the respiratory chain
Most of the time and in most cells, the respiratory chain runs at rates that aresubstantially below the maximal rate. How is the flow through the respiratorychain controlled? In a healthy and not maximally exerted cell, there is muchmore ATP than ADP or phosphate, so that these become limiting for the flow.If ATP synthase is short of substrates, the proton-motive force will not bedissipated, so that the proton pumps will have a harder time to extrude moreprotons and will eventually stall. Since electron transport and proton pumpingare tied to one another, this means that dehydrogenation of NADH and FADH2
will stall as well.The flow rate of the respiratory chain is also coupled to those of the pre-
ceding pathways of glycolysis and the TCA. Such coupling occurs by negativefeedback at various levels:
1. A low flux through the respiratory chain will lead to the accumulationof NADH, which slows down glyceraldehyde-3-phosphate dehydrogenase, pyru-vate dehydrogenase, and the NAD+-dependent isocitrate dehydrogenase.
2. A low consumption of ATP will result in its accumulation to higher levels.Many enzymes, including phosphofructokinase, are inhibited by ATP.
These regulatory mechanisms are reasonably straightforward. There is,however, one remaining mystery. We have already noted that there are twoforms of isocitrate dehydrogenase, one using NAD+ and the other NADP+ asthe cosubstrate. While the NAD+-dependent form is inhibited by NADH andATP, the NADP+-dependent form, which is actually the more highly expressedone of the two, is not subject to such inhibition. This would suggest that it
6.6 Regulation of the respiratory chain 83
might go at full blast even when the demand for ATP is low and NADH ishigh! How, then, is this enzyme prevented from uncontrolled consumption ofisocitrate?
It appears that, at least during times of low demand for ATP, NADP+-dependent isocitrate dehydrogenase is close to equilibrium. This equilibrium issustained by a high mitochondrial level of NADPH, which in turn is maintainedby NAD+/NADPH transhydrogenase. This remarkable protein, wich is locatedin the inner mitochondrial membrane, is both an enzyme and a transporter. Itreduces NADP+ to NADPH at the expense of NADH. As with ATP synthase, theenzyme reaction is coupled to the translocation of protons:
NADH+NADP+ +H+out → NAD+ +NADPH+H+in (6.8)
Figure 6.14 shows how the function of transhydrogenase is integrated withthe function and regulation of the TCA and the respiratory chain.11
When the demand for ATP is low, NADH and the proton-motive force willboth be at high levels, which will cause the transhydrogenase to reduce NADP+
at the expense of NADH. At equilibrium, the NADPH concentration will behigh, which results in near-equilibrium conditions for the NADP+-dependentisocitrate dehydrogenase also. There may, however, be a low net flux within aninteresting futile cycle, which involves the two isocitrate dehydrogenases andthe transhydrogenase (Figure 6.14a). The net effect of this cycle is the influx ofone proton in each round.
On the other hand, when the demand for ATP is high, the proton-motiveforce and the level of NADH will be lower. Under these conditions, the tran-shydrogenase will switch direction, now consuming NADPH to produce moreNADH, which will be consumed at high rate in the respiratory chain. At thesame time, transhydrogenase will work as an auxiliary proton pump, thus di-rectly contributing to the proton-motive force. This will reduce the level ofNADPH, which in turn will topple the equilibrium of NADP+-dependent isoci-trate dehydrogenase in favour of supplying more NADPH, which will help tokeep the entire process going.
Now that is a marvelous piece of engineering by Mother Nature, isn’t? Thisis as close as it gets to intelligent design.
11This is my take on the subject. There is, however, considerable variety of opinion on the roleof this fascinating enzyme.
84 6 The respiratory chain
Isocitrate Ketoglutarate + CO2
NAD+
NADHNADP+
NADPH
H+
H+
respiratory chain
Isocitrate Ketoglutarate + CO2
NAD+
NADHNADP+
NADPH
H+
H+
respiratory chain
mitochondrial matrix
cytosol
a)
b)
Figure 6.14 Function of NADP+/NADH transhydrogenase, and its integration with theTCA and the respiratory chain. a: Function of transhydrogenase in idling mode, whilethe demand for ATP is low. In this situation, the extra-mitochondrial H+ concentrationis high. Protons enter through transhydrogenase and sustain a high concentrationof NADPH, some of which in turn is dissipated by NADP+-dependent isocitrate dehy-drogenase. b: Function of transhydrogenase at times of high demand for ATP. A highthroughput of the respiratory chain causes the extra-mitochondrial H+ concentration todrop. The proton flow through transhydrogenase now reverses, as does the operationof NADP+-dependent isocitrate dehydrogenase. This results in the provision of extraNADH for the respiratory chain, as well as protons for ATP synthesis.
Chapter 7
Gluconeogenesis
The metabolic pathways we have considered so far account for the completeoxidative degradation of glucose. Glucose is the most important substrate ofenergy metabolism for several reasons:
1. Glucose accounts for a large share of all calories in a typical human diet.2. Some cell types, for example erythrocytes, depend entirely on it for all
their metabolic energy. Others, such as nerve cells, only very reluctantly giveup their preference for it, althougy they can adapt to ketone bodies (whichare formed by fatty acid degradation) as alternative substrates in times ofstarvation.
3. Glucose is the major source of NADPH, which is needed for many biosyn-thetic tasks, including fatty acid and sterol synthesis. The pathway that regen-erates NADPH by oxidation of glucose—the hexose monophosphate shunt—willbe discussed later on.
It is plausible then that a pathway should exist that can turn other sub-strates into glucose if the latter is lacking in the diet. In fact, many carnivorousmammals and many humans—and not merely decadent Westerners,think of thetraditional lifestyle of Inuit—live on diets that are very rich in protein but notglucose. Such diets will typically also be rich in fat; however, fatty acids cannotbe converted to glucose in the mammalian metabolism. Amino acids thereforeare the major source of carbon for the synthesis of glucose. The pathway ofglucose synthesis is called gluconeogenesis.
85
86 7 Gluconeogenesis
Figure 7.1 Overview of gluconeo-genesis and its role in humanmetabolism. Lactate and aminoacids that can be converted topyruvate or to TCA intermediatesserve as carbon sources. The con-version to glucose uses the re-versible reactions from glycolysis,and 4 distinct reactions that cir-cumvent the ones from glycolysisthat are irreversible. These re-actions are catalyzed by pyruvatecarboxylase (1), phosphoenolpyru-vate carboxykinase (2), fructose-1,6-bisphosphatase (3) and glucose-6-phosphatase (4).
Pyruvate
Acetyl-CoACO2
Citrate
Isocitrate
α-Ketoglutarate
Succinyl-CoASuccinate
Fumarate
Malate
Oxaloacetate
CO2
CO2
Amino acids
Glucose
Glucose-6-P
Fructose-6-P
Fructose-1,6-bis-P
Dihydroxyacetone-P + Glyceraldehyde-P
1,3-Bis-P-glycerate
3-P-glycerate
P-enolpyruvate
Lactate1
2
3
4
Gluconeogenesis is essentially confined to two organs: The liver and thekidney. Both organs have an ample supply of amino acids: The liver from theintestine via the portal vein, the kidneys because they extract amino acids fromthe considerable volume (~150 l/day) of plasma ultrafiltrate that represents thefirst stage of urine secretion (see section 14.6).
7.1 Reactions in gluconeogenesis
Gluconeogenesis converts pyruvate and oxaloacetate to glucose. It is essentiallya reversal of glycolysis, with workarounds for those reactions of glycolysis thatare energetically irreversible. These four reactions are highlighted in Figure 7.1.
7.1 Reactions in gluconeogenesis 87
a)
b)
c)
NHNH
O
SNH
O
NH NH
O
SR
O
O
OH
O
O
OH
P
O
O
O
ADP
NH N
O
SR
C
O
OH
ATP
Pi
CH3 C
O
C
O
O
CH2 C
O
C
O
O
CH2
C
O
C
O
O
O
O
Enzyme-B
Enzyme-BH+
Biotin-CO2
Biotin
Enzyme
Figure 7.2 The pyruvate carboxylase re-action. a: The enzyme contains a biotincoenzyme covalently attached to a lysineresidue. The long, flexible arm allows thecoenzyme to interact with multiple activesites. b: In the first step of the reaction,biotin is carboxylated. The carboxyl groupis obtained from a bicarbonate ion (topleft) and requires ATP. Carboxyphosphate(top right) is likely formed intermittently.c: In the second step (which occurs at aseparate active site), the carboxyl group istransferred onto pyruvate. This step re-quires abstraction of a proton from thepyruvate methyl group to yield a carban-ion.
The final reaction in glycolysis is the transfer of the phosphate group fromphosphoenolpyruvate (PEP) to ATP. This reaction is irreversible because of thestrongly exergonic nature of the accompanying rearrangement of pyruvate fromthe enol to the keto form (see section 3.4.4). In gluconeogenesis, it takes twoenzymatic steps to turn pyruvate back into PEP: (1) Carboxylation to oxaloac-etate by pyruvate carboxylase (Figure 7.2), and (2) conversion of the latter toPEP by phosphoenolpyruvate carboxykinase (Figure 7.3a).
Pyruvate carboxylase requires biotin as a coenzyme. This coenzyme is
88 7 Gluconeogenesis
flexibly attached to the enzyme (Figure 7.2a) in a manner reminiscent of lipoicacid in pyruvate dehydrogenase (see Figure 5.2), and for a similar reason: Thereaction occurs in separate steps at different active sites, which with somebiotin-dependent enzymes (though not with pyruvate carboxylase) are locatedon separate enzyme subunits.
Biotin serves as an intermediate carrier of a carboxyl group that is generatedfrom bicarbonate, which is equivalent to CO2. Remarkably, therefore, we areable to metabolically fix CO2, just like plants! Before you try to claim Kyototreaty credits for this ability, however, it is necessary to consider that the verysame molecule of CO2 gets released again in the next step. The whole purposeof its transient fixation consists in the facilitation of the subsequent reaction,which is outlined in Figure 7.3a.
7.2 Glucogenic amino acids and gluconeogenesis
So far, we have seen that the TCA serves to completely degrade acetyl-CoA. Inaddition to this degradative function, the TCA also serves to collect the carbonskeletons of several amino acids, which can be converted into different TCAintermediates (Figure 7.1) and from there to oxaloacetate. Other amino acidscan be converted to pyruvate. Since pyruvate can be converted to oxaloacetate,we can summarize that all amino acids that can be converted to any interme-diate of the citric acid cycle or to pyruvate can be utilized for gluconeogenesis.Such amino acids are referred to as glucogenic. Amino acids that cannot beconverted to glucose but can be converted to ketone bodies instead are referredto as ketogenic. We will learn more about both amino acid degradation andketone body formation later.
7.3 Regulation of gluconeogenesis
7.3.1 The need to control futile cycling
Figure 7.3b compares the reactions of the two phosphatases involved in glu-coneogenesis with the two corresponding kinases that operate in glycolysis.What would happen if, for example, hexokinase and glucose-6-phosphatasewere active at the same time? Let’s write it down:
Glucose + ATP ---------------------------------------→ Glucose-6-phosphate + ATP
Glucose-6-phosphate+H2O ---------------------------------------→ Glucose+ phosphate
We can combine these two equations and cancel glucose and glucose-6-phosphate, since they occur on both sides. We obtain:
ATP+H2O ---------------------------------------→ ADP+ phosphate (7.1)
7.3 Regulation of gluconeogenesis 89
a)
b)
N
CH
CH
CH
O N
N
CH
OH
CH
OH
CH2
NH
O
OPO
O
O
PO
O
O
PO
O
O
NH2
CCH2
O
O
OH
P O
OH
O
CCH2
O
O
OH
CCH2
O
O
OH
O
O
CO2
Glucose
Glucose-6-phosphate
Fructose-6-phosphate
Fructose-1,6-bis-phosphate
H2O
phosphate
H2O
phosphate
1
2
3
Figure 7.3 Further reactions in gluconeogenesis. a: PEP carboxykinase uses GTP toconvert oxaloacetate to PEP. The carboxylate group serves to facilitate the transientformation of the enolate anion. b: Fructose-1,6-bisphosphatase (1) and glucose- 6-phosphatase (3) revert the two initial phosphorylations that occur in glycolysis. Theydo no regenerate ATP but simply remove the phosphate groups by hydrolysis, which isan exergonic reaction. 2: Phosphohexoseisomerase.
This means that the simultaneous activity of the two enzymes would causeunrestricted, idle hydrolysis of ATP. A pathway with no other effect than theexpenditure of ATP (or some other form of metabolic energy) is called a futilecycle. Futile cycling will also result from simultaneous activity of of fructose-1,6-bisphosphatase with phosphofructokinase, and of pyruvate kinase withpyruvate carboxylase and PEP carboxykinase, with the latter differing only byhydrolysing GTP instead of ATP.
It is clear that unchecked operation of futile cycles would be a disaster – allATP would be rapidly consumed, and the energy simply be dissipated as heat.Of note, the liver has a temperature well above the body temperature (39-40 ◦C),and a fair share of the process heat generated in liver metabolism is believed
90 7 Gluconeogenesis
to derive from the futile cycles just discussed. Still, there are regulatory mecha-nisms to keep these cycles in check. While this applies to all enzymes involved,as an example we will consider those that concern phosphofructokinase andfructose-1,6- bisphosphatase.
7.3.2 ATP, ADP and AMP in the regulation of phosphofructokinase andfructose-1,6- bisphosphatase
Phosphofructokinase is inhibited by ATP. This makes sense, since ATP forma-tion is the main purpose of glycolysis (in conjunction with the TCA and therespiratory chain). On the other hand, depletion of ATP will result in a buildupof ADP and AMP; it therefore also makes sense that ADP and AMP stimulatephosphofructokinase (section 2.5). While most ATP-consuming reactions yieldADP rather than AMP, AMP is formed from ADP by adenylate kinase:
2 ADP ---------------------------------------→ ATP+AMP
This allows for a makeshift regeneration of some ATP from ADP. Also, if weconsider the equilibrium:
K = [ATP] [AMP]
[ADP]2a [AMP] = K [ADP]2
[ATP]
we see that [AMP] will vary with the square of [ADP]; [AMP] therefore is themore sensitive parameter to detect changes in the availability of ATP.
Fructose-1,6-bisphosphatase is stimulated by ATP and inhibited by AMP.This behaviour is opposite to that of phosphofructokinase, and it ensures thatone enzyme will be active when and only when the other one is inactive, so thatfutile cycling and ATP hydrolysis is avoided.
7.3.3 Hormonal control of phosphofructokinase andfructose-1,6-bisphosphatase
Both enzymes also show opposite responses to another molecule, fructose-2,6-bisphosphate (Figure 7.4b). This molecule is not a regular metabolite of glucosemetabolism but is synthesized solely for the sake of regulation, at levels muchlower than fructose-1,6- bisphosphate. Its concentration is under the controlof hormones via the secondary messenger 3’,5’-cyclo-AMP (cAMP). The entirecascade consists of the following stages (Figure 7.4c):
1. The hormones bind to their respective receptors on the cell surface,which then promote formation (glucagon and epinephrin) or degradation (in-sulin) of cAMP.
2. cAMP binds to and activates protein kinase A, which phosphorylates theenzyme phosphofructokinase 2. Phosphorylation can be reversed by a proteinphosphatase.
7.3 Regulation of gluconeogenesis 91
Fructose-6-P
Fructose-1,6-bis-P
ATP
ADPH2O
Pi
Phospho-fructokinase
Fructose-1,6bisphosphatase
ATP
AMP
Fructose-2,6-bis-P
+
+
+
-
-
-
PO
O
OH
OO
OH
OH
CH2
OH
O
P OH
O
O
CH2
OHO
OH
OH
CH2
O
O
P OH
O
O
CH2
POH
O
O
a)
b)
c)
cAMP
+
+
Epinephrin,glucagon Insulin
Protein kinase A
PFK-2/(bis-P’ase) (PFK-2)/bis-P’ase
ATP ADPP
Fructose-6-P
Fructose-2,6-bis-P
AMPATP
+
Figure 7.4 Regulation of phosphofructokinase and of fructose-1,6-bisphosphatase.a: Allosteric effectors have opposite effects on the two enzymes. b: Structure offructose-2,6-bisphosphate (right). Fructose-1,6-bisphosphate is shown for comparison.c: Control of fructose-2,6-bisphosphate levels by hormones. See text for details.
92 7 Gluconeogenesis
3. Phosphofructokinase 2 has two opposite activities: In the dephospho-rylated state, it acts as a kinase and therefore increases the level of fructose-2,6-bisphosphate. In the phosphorylated state, it acts as the correspondingphosphatase and therefore lowers the level of fructose-2,6- bisphosphate.
So, what is the upshot of these hormone actions? Let’s go over it step bystep again. Here is the effect of glucagon and epinephrin:
Glucagon ↑ or epinephrin ↑ → cAMP ↑ →PFK-2 ↓, fructose-2,6-bisphosphatase ↑ → fructose-2,6-bisphosphate ↓ →
gluconeogenesis favoured
This is the effect of insulin:
Insulin ↑ → cAMP ↓ →PFK-2 ↑, fructose-2,6-bisphosphatase ↓ → fructose-2,6-bisphosphate ↑ →
glycolysis favoured
Thus, glucagon and epinephrine promote gluconeogenesis, inhibit glycolysisand so increase availability of glucose, while insulin has the opposite effects andpromotes the net consumption of glucose. It should be noted that ATP and AMPadjust the activity of phosphofructokinase and fructose-1,6-bisphosphatase ac-cording to the intracellular situation, whereas the hormones, via cAMP andfructose-2,6-bisphosphate, control the same enzymes on behalf of the metabo-lism of the body as a whole. This is a nice example of how diverse regulatorysignals are integrated at the molecular level.
7.4 Energy balance of gluconeogenesis
In the formation of pyruvate from glucose—that is, glycolysis—there was a netgain of two molecules of ATP per molecule of glucose. How many molecules ofATP are required to revert the process?• Going from pyruvate to phosphoenolpyruvate via oxaloacetate costs one
ATP and one GTP, which is equivalent to 2 ATP.• A third ATP is spent in the (reversible) phosphorylation of 3- phospho-
glycerate to 1,3-bisphosphoglycerate by phosphoglycerate kinase.• We need two molecules of pyruvate for one molecule of glucose; this
works out to six molecules of ATP per molecule of glucose.If glycolysis were reversible without change (which it is not), then only two
molecules of ATP would have to be consumed. The expenditure of 4 extramolecules of ATP is necessary to revert the energy balance of the pathway sothat it actually proceeds into the opposite direction. Formation of no more thantwo ATP molecules makes it exergonic to turn glucose into pyruvate, whereasexpenditure of extra ATP makes it exergonic to turn pyruvate back into glucose.
7.5 The Cori cycle 93
glucose
pyruvate lactate
NAD+
pyruvate
glucose
6 ADP
6 ATPNADH+H+
2 ADP
2 ATPNAD+
NADH+H+
Figure 7.5 The Cori cycle. Lactate generated from glucose by anaerobic glycolysis inthe skeletal muscle during exercise is moved to the liver, reoxidized to pyruvate andturned back into glucose by gluconeogenesis, which is then returned to the muscle orother peripheral tissues.
7.5 The Cori cycle
We have seen in section 3.6 that lactate accumulates in anaerobic glycolysis.Cells producing lactate will release it into the blood. What becomes of it subse-quently? It may be taken up by the liver, reoxidized to pyruvate, and fed backinto gluconeogenesis. The combination of glycolysis in peripheral tissues withgluconeogenesis in the liver is referred to as the Cori cycle (Figure 7.5).
While gluconeogenesis is certainly important in the utilization of lactategenerated in the skeletal muscle, most books fail to point out that the twostages of this ‘cycle’ cannot be active at the same time, for the following reasons:
1. Under conditions of maximal exercise, liver perfusion is minimized, andthe liver itself is quite as anaerobic as the muscle itself. It gets even less oxygenthan the muscle does, and without oxygen, the liver cannot regenerate the NAD+
needed for turning lactate into pyruvate any more than the muscle could.2. Since gluconeogenesis costs more ATP than glycolysis generates (section
7.4), the net energy balance of the Cori cycle will be the expenditure, not thegain of ATP – not helpful for sustaining maximal exercise.
So, what really happens is that after maximal exercise has stopped, the liverslowly scoops up the lactate that has accumulated in the blood and turns itback into glucose. Thus, the Cori cycle operates asynchronously rather thancontinuously – it has more similarity with the hog cycle of the agricultural mar-kets than with a regular metabolic cycle such as the TCA. The Cori cycle does
94 7 Gluconeogenesis
indeed run synchronously with cells such as erythrocytes and thrombocytes,which don’t have mitochondria and thus rely completely on anaerobic glycol-ysis even under aerobic conditions. However, their energy requirements areminuscule compared to those of skeletal muscle.
7.6 The glyoxylate cycle
It was stated above that fatty acids cannot be utilized for gluconeogenesis. Also,it was mentioned earlier that fatty acids are degraded via acetyl-CoA. Sinceacetyl-CoA is degraded via the TCA, and TCA intermediates can be utilized forgluconeogenesis, why is it not possible to turn fatty acids into glucose? This isbecause of the different roles of the TCA intermediates in the two processes:
In the degradation of acetyl-CoA, they have a catalytic role – they are re-quired for the cycle to run, but there is no net gain or loss of intermediates.Acetyl-CoA fed into the TCA is consumed without increasing the pool of TCAintermediates. Now, in gluconeogenesis, oxaloacetate is drained from the poolof TCA intermediates. Thus, the TCA can only sustain gluconeogenesis to theextent it is supplied with new intermediates from other sources.
To turn fatty acids into glucose, we therefore need a means to use acetyl-CoA for increasing the pool of intermediates. Such a pathway does not exist inmammals, but it does exist in plants; it is known as the glyoxylate cycle. It is aside-road to the TCA that allows them to use two molecules of acetyl-CoA percycle for the net synthesis of one C4- intermediate. Two reactions are requiredfor this cycle (Figure 7.6):
1. Isocitrate is split into succinate and glyoxylate by isocitrate lyase. Sincethe isocitrate dehydrogenase and the α-ketoglutarate dehydrogenase reactionsare bypassed, the loss of two carbons as CO2 is avoided; these carbons areretained in the form of glyoxylate.
2. Glyoxylate combines with the second acetyl-CoA to form one molecule ofmalate. This reaction is catalysed by malate synthase, and like the citrate syn-thase reaction it is pushed forward by the concomitant hydrolysis of coenzymeA.
You know that many plant seeds are very rich in oil (=fat). The glyoxylatecycle enables plant seeds to store metabolic energy and carbon as fat, and touse it for the synthesis of glucose and other carbohydrates during germination.Fat is water-free and has approximately twice the content of metabolic energyper gram of carbohydrates; it is therefore both more compact and also moreresistant to microbial degradation than starch.
7.7 Additional roles of gluconeogenetic enzymes
Two enzymes that occur in gluconeogenesis have important roles in othermetabolic pathways. One of them is glucose-6-phosphatase. Glucose-6-phosphate
7.7 Additional roles of gluconeogenetic enzymes 95
CH2 COOH
CH COOH
CH
COOHOH
CH2 COOH
CH2
COOH
CH
COOHO
CH
COOHO
CH3 C
O
S CoA CH2 C
O
OH
CH
COOHOH
SH CoA
Isocitrate lyase
Malate synthase+
Citrate
Isocitrate
Succinate
Fumarate
Malate
Oxaloacetate
Acetyl-CoA
Glyoxylate
Acetyl-CoA
Glucose
a)
b)
Glyoxylate
1
2
Figure 7.6 The glyoxylate cycle of plants. a: Two enzyme reactions are required inaddition to those of the TCA. Both reactions are similar to the citrate synthase reaction.b: Overview of the cycle. Dotted arrows represent reactions of the TCA that are skippedby the glyoxylate cycle. (The conversion of oxaloacetate to glucose has been abridged.)
can be generated not only by gluconeogenesis but also during degradation ofglycogen, the polymeric storage form of glucose (see chapter 8). Glucose-6-phosphate released from glycogen likewise must undergo dephosphorylationto glucose by glucose-6-phosphatase before release into the bloodstream.
The other enzyme that deserves mention is pyruvate carboxylase. If youlook at Figure 1.2, you will see that pyruvate can enter the TCA via two routes:As a substrate, after conversion to acetyl-CoA, and as a C4-intermediate, namelyoxaloacetate. If a shortage of TCA intermediates occurs, acetyl-CoA will backup. Acetyl-CoA allosterically activates pyruvate carboxylase, which producesoxaloacetate and helps restore the level of TCA intermediates back to normal.
Pyruvate carboxylase can also help to provide NADPH, which otherwise isprovided prominently by the hexose monophosphate shunt. Oxaloacetate is
96 7 Gluconeogenesis
reduced to malate by malate dehydrogenase—as we have seen, this reactionoccurs both in mitochondria and in the cytosol—and malate is then cleaved bymalic enzyme, which yields pyruvate and NADPH. This reaction is consideredin more detail in section 12.1.
Chapter 8
Glycogen metabolism
Glycogen is a polymeric storage form of glucose (Figure 8.1). It is very similarto amylopectin, the branched polyglucose molecule found in starch (Figure 1.6);the only difference is that glycogen is more highly branched. It is synthesizedfrom glucose in times of plenty, that is after a meal rich in carbohydrates, andconverted back to glucose when the later is in demand. It is found in manytissues, but only two of these are quantitatively important:
1. The liver stores glycogen for the purpose of releasing the glucose con-tained in it into the circulation; glycogen stored here therefore serves the entireorganism. The glycogen content of a fully stocked liver amounts to as much as10% of its wet weight, that is about 150-200 grams. Since glycogen is so similarin structure to starch, this is comparable to 200 grams of dry spaghetti.
2. According to the books, the skeletal muscle stores glycogen only for itsown use – glucose released from glycogen in the muscle is not released into theblood but consumed then and there. The concentration of glycogen in muscletissue is lower than in the liver, but because of the large mass of sceletal musclethe amount of glycogen stored in muscle is actually about twice that found inthe liver.
The alleged inability of skeletal muscle to release glucose would result froma lack of the enzyme glucose-6-phosphatase, without which free glucose cannotbe obtained from glycogen. However, recent work has shown that this enzymeis indeed expressed at appreciable levels in the muscle also.1 This raises the
1See Shee et al., J Biol Chem 279:26215-9 (2004) and references cited therein.
97
98 8 Glycogen metabolism
GlycogeninO-TyrO O O O O OO
O O O OO O
CH2
O O O O O
CH2
O O O O O
CH2
Figure 8.1 Structure of glycogen: A single α-1→4-polymer of glucose is attached tothe protein glycogenin. Additional polymers branch of from the first one and fromother branches. Two adjacent branching points are not 2 (as shown here) but 6-8glucose residues apart. Attachment of the branches is by α-1→6-glycosidic bonds. Thetotal number of glucose residues in the entire polymer can be as high as 105.
interesting possibility that, contrary to traditional belief, skeletal muscle doesfunction as a reservoir of glucose. This is important, since an accurate un-derstanding of blood glucose regulation and homeostasis is essential in thetreatment of diabetes mellitus.
Why do organisms store glucose in polymeric rather than in free form? Freeglucose would cause an inacceptably high osmotic pressure inside the cell. Theosmotic pressure associated with a solute follows the gas equation:
pV = nRT a p = nVRT
This means that the osmotic pressure is proportional to the number ofmolecules per volume, or the molar concentration. Consider the amount ofglycogen stored in the liver: 10% equals 100 g/l. Each glucose residue in glycogenhas a molar weight of 162 Da, which works out to roughly 0.6 mol/l glucoseresidues. This is approximately twice as high as the total concentration of smallsolutes inside the liver cell. Therefore, if all the glycogen were converted toglucose, the osmotic pressure would triple, and the liver cell would suck waterlike a delirious camel and burst. Polymerization of glucose vastly reduces itsmolar concentration and therefore its osmotic activity; it makes storage of largeamounts of glucose ‘bio-compatible’.
8.1 Glycogen synthesis
The following enzyme reactions occur in glycogen synthesis (Figure 8.2):1. Glucose activation,2. Initiation of glycogen synthesis,
8.1 Glycogen synthesis 99
3. Chain elongation,4. Introduction of branch points.
8.1.1 Glucose activation
Glucose activation consists in the formation of UDP-glucose from glucose-6-phosphate, which to this end is converted to glucose-1-phosphate by the en-zyme phosphoglucomutase. Glucose-1-phosphate is then activated to UDP-glucose by glucose-1-phosphate uridylyltransferase; this reaction uses uridinetriphosphate (UTP) and releases pyrophosphate.2
8.1.2 Initiation and elongation
The first molecule of glucose is attached to the OH group of a tyrosine residuein the small protein glycogenin, which therefore serves as a seed for glycogensynthesis. Subsequently, another glucose subunit is attached to the 4-OH groupof the first one, and this process is then repeated over and over, giving rise to along, linear polysaccharide. UDP-glucose is the substrate in both the initiationstep and the repetitive chain elongation steps. At this point, all glycosidicbonds in the polysaccharide are in the α-1→4 configuration, so that this stage ofnascent glycogen resembles amylose. The enzyme responsible for the initiationand extension of the linear polymer is glycogen synthase.
It is interesting to note that the carbon 1 of each glucose subunit is in theα-configuration throughout, both in UDP-glucose, and in glycogen. What doesthis tell us about the mechanism of the reaction? Nucleophilic substitutionscan occur either synchronously or asynchronously. In the first case, which iscalled the SN2 mechanism, one substituent leaves as the other arrives; each ofthem holds on with ‘half a bond’ to the transition state.
As a result, the new substituent will occupy a position opposite to theold one. With asymmetric C atoms, this gives rise to the so-called Waldeninversion. Since no such inversion occurs in glycogen chain elongation (noconversion from α- to β-configuration; Figure 8.3), it follows that this cannot bethe correct mechanism. Instead, the first substituent (UDP) leaves, taking bothbond electrons with it. The carbon remains as a carbocation with three ligandsonly. The enzyme then directs the new substituent—the terminal glucose ofthe growing glycogen chain—to attack from the same side the UDP left. This istherefore an SN1 reaction mechanism.3
2Whereever pyrophosphate is released, it is subsequently cleaved to two phosphate ions bypyrophosphatase. This cleavage is exergonic and keeps the concentration of pyrophosphatelow, which in turn makes the first reaction more exergonic. Release of pyrophosphate thereforeprovides a stronger driving force to a reaction than release of monophosphate.
3With non-enzymatic SN1 reactions, we would expect racemization to occur at this stage, asthe new substituent could attack the planar transition state with equal ease from either side.Enzymes, however, can impose a defined route of attack.
10
08
Gly
co
gen
met
abo
lism
Glucose-6-P
Glucose-1-P
O
OH
OH
OH
OH
C
OP
OH
O
O
O
O
OH
OH
OH
CH2OH
P O
O
OH
N
NH
O
O
OHOH
COPO
O
O
PO
O
O
PO
O
O
O
O
O
OH
OH
OH
CH2OH
P O
O
OH
N
NH
O
O
OHOH
COP
O
O
O
P-Pi2 Pi
GlycogeninOH
O
O
OH
OH
OH
CH2OH
P O
O
OH
N
NH
O
O
OHOH
COP
O
O
O
O
OH
OH
OH
CH2OH
O Glycogenin
N
NH
O
O
OHOH
COPO
O
O
POH
O
O
O
UDP
Tyr
Tyr
GlycogeninO-TyrHO O O O O O
UDP-glc UDP-glc UDP-glc UDP-glc
UDP UDP UDP UDP UDP
UDP-glc
a)
b)
c)UTP
UDPglucose
GlycogeninO-TyrO O O O OOO
CH2OH
GlycogeninO-TyrHO O O O O
CH2
HO O O O
d)
Figure 8.2 Reactions in glycogen synthesis (1). a: Activation of glucose. Glucose-6-phosphate is converted to glucose-1-phosphateby phosphoglucomutase. Glucose-1-phosphate uridylyltransferase converts glucose-1-phosphate to UDP-glucose. Pyrophosphatereleased is cleaved by pyrophosphatase. b: 1 molecule of UDP- glucose reacts with glycogenin. c: Glycogen synthase linearlypolymerizes glucose residues, again using UDP-glucose as a substrate. d: Branching enzyme transfers the end of a growing chainonto the C6 atom of an internal glucose residue. Both C4 ends are subsequently extended by glycogen synthase until branchingrepeats.
8.2 Glycogen degradation 101
8.1.3 Branching enzyme
Branching enzyme cuts a string of glucose residues from a growing end andgrafts it onto the sixth C atom of a glucose residue within a chain (Figure 8.2).Branching can be repeated, such that the distance between adjacent branchpoints will be 6-8 glucose residues, and branches will carry other branches inturn. What is the purpose of this? Branching increases the number of free endsfor attachment or—during degradation—removal of single glucose residues.The higher number of branches in glycogen relative to amylose and amylopectinis in line with the fact that animals have a higher metabolic turnover thanplants.
8.2 Glycogen degradation
Two enzymes collaborate in glycogen degradation: Phosphorylase and de-branching enzyme. All glucose subunits that are joined by α-1→4-glycosidicbonds—that is, those in the straight segments—will be released by glycogenphosphorylase. While the most common way of cleaving glycosidic bonds inmetabolism consists simply in hydrolysis, phosphorylase uses phosphate ionsinstead of water to cleave these bonds (Figure 8.4). While this is not an ex-ergonic reaction under standard conditions, it works here because the freeconcentration of glucose-1-phosphate in the cell is low. Glucose-1-phosphateis converted back to glucose-6-phosphate. In the liver, which stores glycogenfor the benefit of the entire body, the lion’s share will be dephosphorylated byglucose-6-phosphatase and released into the circulation. As pointed out above,the same may happen in muscle; however, muscle certainly uses glycogen to alarge extent for its own keepup, and therefore glucose-6-phosphate will oftenbe funneled straight into glycolysis.
Glycogen phosphorylase only degrades the chain ends to within 4 residuesof a branching point. Then, debranching enzyme takes over and transplants thestub to another free end. However, it leaves behind a single residue attachedby a α-1→6-glycosidic bond, which it subsequently cleaves by hydrolysis andreleases as glucose (Figure 8.4b).
8.3 Glycogen storage diseases
While the above reactions should account for the complete degradation ofglycogen (except for the very last glucose residue attached to glcyogenin), thereis more than meets the eye. This is evident from the fact that a genetic defectin an apparently unrelated enzyme—lysosomal maltase,4 which cleaves the α-1→4-glycosidic bond between two glucose residues—causes the accumulation
4The reaction is the same as catalyzed by the brush border maltase in the degradation ofamylose; however, the lysosomal enzyme is a separate entity and has an acidic pH optimum, inkeeping with the acidic environment inside the lysosomes.
102 8 Glycogen metabolism
O
O
OH
OH
OH
CH2OH
UDP
HO
O
OH
OHO
CH2OH
Glycogen
O
C+
OH
OH
OH
CH2OH
H
O UDP
O
OH
OH
OH
CH2OH
H O
O
OH
OHO
CH2OH
Glycogen
a)
b)
O
O
OH
OH
OH
CH2OH
UDP
H
O
O
OH
OHO
CH2OH
Glycogen
O
O
OH
OHO
CH2OH
Glycogen
O
OH
OH
OH
CH2OH
H
O
O
OH
OHO
CH2OH
Glycogen
O
OH
OH
OH
CH2OH
H
O UDP
+UDP
Figure 8.3 Reaction mechanism of glycogen synthase. a: A SN 2 mechanism shouldresult in a Walden inversion, converting the α-glycosidic bond in UDP-glucose to a β-glycosidic bond, which is not observed. b: A SN 1 mechanism is compatible with theretention of the α-configuration.
of glycogen in all kinds of tissues, with particularly detrimental consequences inheart muscle. The accumulation of glycogen pudding inside the cell interfereswith cell motility and therefore with the contraction of the heart, and heartfailure leads to death. The condition is known as Pompe’s disease.
Other enzymes may be deficient as well. A deficiency of phosphorylase inthe muscle is known as McArdle’s disease; patients suffer from rapid exhaus-tion and muscle pain during exertion. A defect of glucose-6-phosphatase ismostly manifest in the liver and kidney. It will affect the release of glucosefrom glucose-6-phosphate formed by both glycogen degradation and gluconeo-genesis, which in turn leads to critically low blood glucose (hypoglycemia).
8.4 Regulation of glycogen metabolism 103
Pi
glc-1-P
glc–glc–glc–glc–glc–glc–glc–glc
Pi
glc-1-P
glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc
glc–glc–glc–glc–glc–glc–glc–glc–glc–glc
glc–glc–glc–glc
glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc
glc
glc
glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc–glc
a)
b)
Figure 8.4 Degradation of glycogen. a: Glycogen phosphorylase cleaves α-1→4-glycosidic bonds using phosphate ions to yield glucose-1-phosphate. Phosphorylasestops 4 glucose residues shy of a branching point. b: Debranching enzyme cleaves allbut one residue off a branch and adds them to another free end, which can then again bedegraded by phosphorylase. The remaining single residue (held by a α-1→6-glycosidicbond) is removed by hydrolysis.
8.4 Regulation of glycogen metabolism
We have seen before that phosphofructokinase and the complementary enzymefructose-1,6-bisphosphatase are regulated by both intracellular and extracellu-lar signals. The same applies to the enzymes involved in glycogen metabolism.
The allosteric regulatory effects by ATP, AMP and glucose-6-phosphate (Fig-ure 8.5a) make sense: Depletion of ATP would be an excellent reason to regener-ate it by tapping into the glucose store. On the other hand, glucose-6-phosphatewill be plentyful when glucose itself is abundant, so it should promote glycogensynthesis rather than breakdown. Hormonal control (Figure 8.5b) is similar tothat of gluconeogenesis (section 7.3): Protein kinase A decreases glycogen syn-thesis via direct phosphorylation of glycogen synthase. Glycogen breakdownis stimulated by phosphorylation of a dedicated enzyme, phosphorylase kinase,which then in turn phosphorylates glycogen phosphorylase. Note that glycogen
104 8 Glycogen metabolism
Figure 8.5Regulation ofglycogen metabo-lism. a: Allostericcontrol of glycogensynthase and ofphosphorylase. b:Hormonal control.See text for details.
Glycogen
Glucose-1-P
UDP-glucose
phosphorylaseglycogen synthase ATP -
AMP +
Glc-6-P+ -
Glycogen synthase (active)
Glycogen synthase–P (inactive)
Phosphorylasekinase (inactive)
Phosphorylasekinase–P (active)
Phosphorylase(inactive)
Phosphorylase-P(active)
Protein kinase A
cAMP
+
+
Epinephrin, glucagon Insulin
AMPATP
+
a)
b)
synthase and phosphorylase respond in opposite ways to phosphorylation: Thefirst one is inactivated, the second activated.
There are regulatory differences between glycogen phosphorylase in muscleand liver: Glucose inhibits the liver enzyme but not the muscle enzyme, andCa++ stimulates the muscle enzyme but not the liver enzyme. Recall that Ca++
is also the trigger for muscle contraction, so it seems that the simultaneousstimulation of glycogen breakdown occurs in anticipation of increased ATPrequirement. This is an example of the usefulness of isozymes, that is enzymesthat catalyze the same reaction yet are different molecules, and therefore canpossess different regulatory adaptations.
Chapter 9
The hexose monophosphate shunt
We are (finally) nearly done with glucose-6-phosphate. Figure 9.1 summarizesthe various pathways that it is part of, and I hope that by now you recognizemost of them. The final one of these pathways to be covered in this class isthe hexose monophosphate shunt. Since both pentoses and hexoses—as wellas trioses, tetraoses, and heptoses—occur in it, this pathway is sometimes alsoreferred to as the ‘pentose phosphate pathway’.
A single passage of glucose-6-phosphate through the hexose monophos-phate shunt oxidizes it to the C5-sugar ribulose-5-phosphate, releasing onemolecule of CO2. In the process, two molecules of hydrogen are transferred toNADP+, yielding NADPH. The ribulose-5-phosphate can be turned into ribose-5-phosphate and then used for the biosynthesis of nucleotides. Alternatively, itcan be fully oxidized to yield more CO2 and NADPH (Figure 9.2). The hexosemonophosphate shunt therefore provides a second means for complete degra-dation of glucose to CO2, apart from the glycolysis / TCA pathway we haveseen before. However, the purpose of this second oxidative pathway consistsnot in the regeneration of ATP but in the formation of NADPH. This coenzymeis required in many biosynthetic reactions, some of which we will consider be-low. Glycolysis and TCA don’t fill this need, because all hydrogen they abstractaccumulates as NADH or FADH2.1
1Note, however, that there are some auxiliary pathways that can convert NADH to NADPH; oneof them is discussed in section 7.7.
105
106 9 The hexose monophosphate shunt
Glucose-6-phosphate
Fructose-6-phosphate
Glucose
Glucose-1-phosphate
Glycogen
Ribulose-5-P + CO2 + 2 NADPH
6 CO2 + 6H2O
6-P-Gluconolactone
Glucose and other sugars as elements of polysaccharides, glycolipids, glycoproteins
Pyruvate
UDP-Glucose
Figure 9.1 Metabolic fates of glucose-6-phosphate. The hexose monophosphateshunt is indicated by underlining.
Dehydrogenation of pyruvate and the TCA occur in the mitochondria, whichis useful because the NADH generated is then fed into the respiratory chain. Incontrast, the hexose monophosphate shunt occurs entirely in the cytoplasm.This is in keeping with the fact that most of the biosynthetic reactions involvingNADPH also occur in the cytoplasm (or in the ER, which is still outside themitochondrion).
9.1 Reactions in the hexose monophosphate shunt
The reactions occurring in the hexose monophosphate shunt can be dividedinto two phases (Figure 9.2a): (1) Oxidation of glucose-6-phosphate to ribu-lose-5-phosphate, and (2) regeneration of glucose-6-phosphate from ribulose-5-phosphate.
The two phases may operate at the same time, or independently from eachother. While glucose oxidation is irreversible, the interconversion of glucose-6-phosphate to pentose phosphates is reversible (Figure 9.2b). With a typicaldiet, reasonably rich in starch, the net flow of sugar conversion will be fromhexoses to pentoses. However, when eating meat only, our intake of ribose inthe form of RNA will be a very significant fraction of the total carbohydrates,and the net flow in the hexose monophosphate shunt will likely go the otherway (Figure 9.2b, bottom).
9.1 Reactions in the hexose monophosphate shunt 107
a)
b)
Glucose-6-P
2 NADP+
2 NADPHCO2
Ribulose-5-P
Regenerate 5Glucose-6-Pfrom 6 Ribulose-5-P
Ribose-5-P
biosynthesisof nucleotides and nucleic acids
Glucose-PPentose-P NADPH
CO2
Glucose-PPentose-P NADPH
Glucose-PPentose-P NADPH
Figure 9.2 Overview of the hexose monophosphate shunt. a: Glucose-6-phosphateis oxidized and decarboxylated to ribulose-5-phosphate. The hydrogen abstracted istransferred to NADP+. The ribulose-5-phosphate can be converted back to a propor-tionally dimished amount of glucose-6-phosphate, or it can be diverted for the purposeof nucleotide synthesis. b: The net flow of metabolites through the hexose monophos-phate shunt may vary depending on the metabolic situation.
9.1.1 Reactions in the oxidative stage
Three enzymes are required for the oxidative phase (Figure 9.3):1. Glucose-6-phosphate dehydrogenase reduces one molecule of NADP+ to
NADPH and produces 6-phosphogluconolactone.2. Gluconolactonase cleaves the internal ester bond and produces 6- phos-
phogluconate.3. 6-Phosphogluconate dehydrogenase reduces another molecule of NADP+
and decarboxylates 6-phosphogluconate to the pentose ribulose-5-phosphate.After completion of the oxidative phase, NADPH generation is over, and
everything that remains is juggling sugars in order to regenerate hexoses frompentoses.
9.1.2 Reactions in the sugar juggling phase
It is important to note that the formation of hexoses from pentoses is notstoichiometric for the sugar molecules. Instead, it is stoichiometric for thecarbons within them: 6 molecules of the C5 compound ribulose-5-phosphateare converted to 5 molecules of the C6 compound glucose-6-phosphate.
108 9 The hexose monophosphate shunt
O
OH
OH
OH
OH
O P
O
OH
O
OH
NADP+
NADPH+H+
H2O
O
OOH
OH
OH
C
OP
OH
O
O
6-P-Glucono-lactone
6-P-Gluconate
1
NADP+
NADPH+H+
CO2
3
O
OH
OH
OH
OH
C
OP
OH
O
O
Glucose-6-P
OH
O
OH
OH
O P
O
OH
O
Ribulose-5-P
Nucleotides
2
Figure 9.3 The oxidative stage of the hexose monophosphate shunt. Enzymes:1: glucose-6- phosphate dehydrogenase; 2: 6-phosphoglucolactonase; 3: 6-phosphogluconate dehydrogenase.
The reactions in the second phase of the hexose monophosphate shunt aredepicted in Figure 9.4:
1. Two molecules of ribulose-5-phosphate are converted to xylulose-5- phos-phate and to ribose-5-phosphate by ribulose-5-phosphate epimerase and ribu-lose-5-phosphate isomerase, respectively.
2. Transketolase transfers a C2 unit from the xylulose-5-phosphate to theribose-5-phosphate, yielding glyceraldehyde-3-phosphate and the C7 sugar se-duheptulose-7-phosphate.
3. Transaldolase transfers a C3 unit from the seduheptulose-7-phosphateback to glyceraldehyde-3-phosphate, yielding fructose-6-phosphate and the C4
sugar erythrose-4-phosphate. The fructose-6-phosphate may enter glycolysis,or may be converted back to glucose-6-phosphate by phosphohexose isomerase.
4. Transketolase transfers a C2 unit from another molecule of xylulose-5-phosphate to erythrose-4-phosphate. This yields a second molecule of fructose-6-phosphate and again glyceraldehyde-3-phosphate. Both may enter glycolysisor be converted back to glucose-6-phosphate.
The conversion of glyceraldehyde-3-phosphate back to glucose-6-phosphatevia fructose-1,6-bisphosphate utilizes the enzymes from gluconeogenesis andglycolysis that you so fondly remember from the previous chapters. Recallthat aldolase needs two molecules of triose phosphate to form one molecule offructose-1,6- bisphosphate. This results in an overall stoichiometry of 2.5 mo-lecules of hexose per 3 molecules of pentose, or five hexoses per six pentoses.
9.1 Reactions in the hexose monophosphate shunt 109
O
OH
OH
OH
O P
OH
C6
O
OH
OH
O P
C4
OH
OH
OH
OH
O P
OH
O
C7
O
OH
O P
C3
OH
O
OH
OH
O P
C5
O
OH
OH
OH
O P
C5
O
OH
OH
OH
O P
OH
C6C5
OH
O
OH
OH
O P
OH
O
OH
OH
O P
C5
RI
RETK
TA
OH
O
OH
OH
O P
C5
OH
O
OH
OH
O P
C5
O
OH
O P
C3
GNRE
O
OH
OH
OH
O P
O P
0.5 C6
TK
Figure 9.4 The glucose-6-phosphate regeneration phase. Enzymes: RE, ribulose-5-phosphate epimerase; RI, ribulose-5-phosphate isomerase; TK: Transketolase; TA:Transaldolase; GN, Enzymes from gluconeogenesis. Braces indicate the C2 and C3 unitstransferred by transketolase and transaldolase, respectively.
The juggling of sugar chain length depicted in Figure 9.4 is brought aboutby two enzymes, transaldolase and transketolase. While the sugar substratesthey act upon appear to be quite varied at first glance, they fall into just twostructural classes, the members of each of which differ only in chain length(Figure 9.5). The two enzymes only interact with the topmost parts of thesesugar molecule, so that the chain length of the remainder doesn’t enter intothe picture and does not create a need for separate enzymes. The basic idea ofchain length variation is that transketolase always transfers two-carbon units,whereas transaldolase always transfers three-carbon units. Sequential reaction
110 9 The hexose monophosphate shunt
Fructose-6-P Seduheptulose-7-P
Ribulose-5-P
OH
O
OH
OH
O P
Xylulose-5-P
OH
O
OH
OH
O P
O
OH
OH
OH
O P
OH
OH
OH
OH
OH
O P
OH
O
Erythrose-4-PRibose-5-P Glyceraldehyde-3-P
O
OH
O P
O
OH
OH
O P
O
OH
OH
OH
O P
Ketoses
Aldoses
Figure 9.5 The intermediates of the glucose-6-phosphate regeneration phase formtwo homologous series of ketoses and aldoses, respectively. Transketolase and transal-dolase cleave C2 and C3 units, respectively, from the ketoses and graft them onto thealdehyde group of the aldoses.
of the two enzymes will therefore lead to a net change of the substrate chainlength by one carbon.
9.2 Mechanisms of transketolase and transaldolase
The mechanisms of transketolase and transaldolase are depicted in Figure 9.6.Transketolase employs the coenzyme thiamine pyrophosphate, which we en-countered before in the pyruvate dehydrogenase E1 enzyme. As in the pyruvatedehydrogenase reaction, its main function is to provide a carbanion, whichreacts with a carbonyl group and cleaves the adjacent C−C bond, yielding acovalent bond between the coenzyme and the substrate. However, here the sim-ilarity ends. In the second part of the reaction, another aldose substrate entersand carries the transiently coenzyme-bound C2 subunit away. The mechanismof the second half-reaction is exactly the reversal of the first part.
Transaldolase also forms a covalent intermediate with the fragment of thesugar molecule it transfers, and once more the carbonyl bond serves as thepoint of attack for cleavage. However, transaldolase cleaves the bond after thesecond carbon atoms, resulting in the transfer of a C3 unit. The two stages ofthe reaction are again reversals of each other, with the exception that sugarsubstrates of different length will participate.
Considering their mechanisms, it makes sense that both the transketolaseand the transaldolase reactions are readily reversible. So are the isomerasereactions that interconvert the various pentose phosphates. This pathway
9.3 Why do we need both NADH and NADPH? 111
therefore can go either way and bring about the interconversion of pentoses,hexoses and sugars of other lengths as needed.
9.3 Why do we need both NADH and NADPH?
Why NADH and NADPHdifferent roles of is NADPH needed in addition to NADH?The two coenzymes only differ by one phosphate group, and that is far awayfrom where the action is: The redox-active group is the nicotinamide moiety,whereas the adenosine moiety is the one that carries the extra phosphate inNADP (Figure 3.7). While the phosphate group does not make any differenceto the redox chemistry performed by the two coenzymes,2 it enables them tointeract with different sets of enzymes. Consider that all enzymes that consumeor regenerate NAD+ will share the same pool of the coenzyme, and the reactionequilibria of all of them will be affected by the same ratio of oxidized overreduced form ([NAD+]/[NADH]).
While NADP completely resembles NAD in its redox chemistry, the extraphosphate group allows NADP to interact with a different set of enzymes,which does not intersect with the NAD-specific set. Therefore, because thecoenzymes participate in different sets of equilibria, they can themselves bemaintained in different redox states. To use a simile: The two coenzymes arelike to different currencies – both are money, but it is possible to tune the costof borrowing to different economic conditions. Inside the cell, NAD is mostlyoxidized. The ready availability of NAD+ will help to speed up the oxidativereactions in the TCA and glycolysis. In contrast, NADP is mainly found in thereduced state, which will promote reductive reactions in biosynthesis.
Apart from the reaction rates, the free energy (∆G) of the redox reactionswill also be affected by the “choice” of either NAD or NADP as the cosubstrate.Using values of 0.001 for the ratio of [NADH]/[NAD+] and of 100 for [NADPH]/[NADP+],and assuming ∆G0 to be the same for both, this difference works out to about30 kJ/mol.3
9.4 Alternative sources of NADPH
Not all cells that require large amounts of NADPH have a high activity of thehexose monophosphate shunt. Some alternative mechanisms that can provideNADPH are:
1. The combination of pyruvate carboxylase, malate dehydrogenase, andmalic enzyme, which generates NADPH from NADH at the expense of ATPhydrolysis (see section 12.1).
2If we use the same concentrations of reduced and oxidized forms in vitro, there is practicallyno difference in their redox potentials, i.e. they have the same standard redox potentials.
3This ∆G is similar to that provided by the hydrolysis of ATP to ADP. For the cytosol, thissounds about right (see section 9.4 below). It is probably less in mitochondria, where NADH andNADPH are distinguished only by the energy of a single proton transfer (via transhydrogenase).
112 9 The hexose monophosphate shunt
S
N+
R
CH3
R
CH2
C
OH
OH
CH
CH
CH
O
OH
OH
CH OH
CH2 O P
H+
CH O
CH OH
CH2 O P
S
N+
R
CH3
R
CH2
C
OH
O
CH
CH
CH
OH
OH
OH
CH OH
CH2
O P
H
+ H+
C
S
N+
R
CH3
R
CH2
C
CH
OH
O
OH
CH OH
CH2 O P
H+
S
N+
R
CH3
R
CH2
C
CH
OH
OH
O
CH OH
CH2 O P
H
CH
CH
CH
OH
OH
OH
CH OH
CH2 O P
CH2 OH
O
CH2
C
OH
O
CH
CH
CH
OH
OH
OH
CH OH
CH2 O P
E Lys NH2 E Lys NH
+
CH2
C
OH
CH
CH
CH
OH
O
OH
CH OH
CH2 O P
H
E Lys NH
CH2
C
OH
CHOH
CH
CH
O
OH
CH OH
CH2 O P
E Lys NH
+
CH2
C
OH
CH
CH
CH
OH
O
OH
H
CH2 O P
E Lys NH2
CH
CH
O
OH
OH
CH OH
CH2
O P
CH2 OH
H2O H+
H2O
E Lys NH
CH2
C
OH
CHOH
CH
CH
O
OH
CH2 O P
H+
a)
b)
Figure 9.6 Mechanisms of transketolase and transaldolase. a: Transketolase. Onlythe thiazole ring of the thiamine pyrophosphate is shown. The formation of the carban-ion is as described before for pyruvate dehydrogenase (see Figure 5.4). b: Transaldolase.The –N=C– group that links the substrate to the enzyme in the covalent intermediate isa ketimine or Schiff base.
9.5 Uses of NADPH 113
2. NADP+-dependendent isocitrate dehydrogenase, which occurs alongsidethe NAD+-dependent enzyme in mitochondria (see chapter 5) but also in thecytosol. This mechanism seems to be particularly important for fatty acidsynthesis in the liver and the fat tissue (see chapter 10).
9.5 Uses of NADPH
So, where does the NADPH generated in the hexose monophosphate shunt go?Here are several destinations:
1. Synthesis of fatty acids,2. Synthesis of cholesterol,3. Fixation of ammonia by glutamate dehydrogenase,4. Oxidative metabolism by cytochrome P450 enzymes:
(a) Generation of catecholamine mediators (dopamine, epinephrine andnorepinephrine),
(b) Drug metabolism,5. Generation of nitric oxide and reactive oxygen species by phagocytes,6. Scavenging of reactive oxygen species that form as byproducts of oxygen
transport and of the respiratory chain.Topics 1–3 are going to be covered in detail in subsequent chapters. Here,
we will have a brief look at topics 4–6.
9.5.1 Cytochrome P450 enzymes and drug metabolism
Cytochrome P450 enzymes split molecular oxygen (O2) and use NADPH to re-duce one of the atoms to water. The other oxygen atom is retained in a highlyreactive form and utilized to force some kind of reaction on an intrinsicallyreluctant substrate. This is a large class of enzymes that serves a wide vari-ety of metabolic tasks; examples are hydroxylations and epoxydations in thesynthesis of catecholamine and steroid hormones. Cytochrome P450 enzymesalso have a prominent place in the metabolic elimination of drugs. While thereare multiple types of reactions in drug metabolism, one important exampleis the hydroxylation of aliphatic or aromatic moieties in drug molecules. Thehydroxyl group introduced can then be conjugated with hydrophilic groups,which facilitates elimination in the urine (Figure 9.7).
9.5.2 Production of reactive oxygen species
While cytochrome P450 enzymes utilize reactive oxygen in a controlled, con-tained fashion, there are other enzymes that generate reactive oxygen speciesto release them within or outside the cell, for the purpose of killing pathogenicmicrobes. This happens in macrophages and neutrophile granulocytes. Reac-tive oxygen species include the superoxide anion ( ·O−O – ), hydrogen peroxide(H2O2), and hypochlorite (H−O−Cl; this is also the active constituent of bleach).
114 9 The hexose monophosphate shunt
NH
NH
O
O
O
C2H5
NH
NH
O
O
O
C2H5
OH
NADP+
NADPH+H+
O2 H2O
O
O
OH
OH
OH
CO O
UDP
O
OH
OH
OH
CO O
NH
NH
O
O
O
C2H5
O
UDP
Hydrophilic drug molecule
Kidney
Urine
Hydrophobic drug molecule
Liver
More hydrophilic metabolite
a)
b)
Figure 9.7 Role of NADPH in drug metabolism. a: Overview: Drug metabolism occursin the liver and often serves to convert hydrophobic drug molecules to hydrophilicmetabolites, which facilitates excretion in the urine. b: Example: NADPH- dependenthydroxylation of the drug phenobarbital. Hydroxylation is followed by conjugationwith glucuronic acid, which is derived from UDP-glucuronic acid.
The enzyme reactions involved in their formation are summarized in Figure9.8a. NADPH oxidase is responsible for the initial formation of superoxide,from which the other intermediates are derived.
The enzymes are contained in small vesicles called peroxisomes, which willfuse with phagosomes that contain ingested microbes. The reactive oxygenspecies (ROS) generated have an important role in the destruction of the mi-crobes. This is shown by the fact that enzyme defects in the formation of ROScause severe immunodeficiencies, in particular with respect to bacterial infec-tions. ROS also have an important share in the tissue destruction that occursin inflammation.
9.6 Glucose-6-phosphate dehydrogenase deficiency 115
NADPH oxidase
2 O2 + NADPH 2 O2- + NADP+ + H+
2 O2- + 2 H+ H2O2 + O2
Superoxide dismutase
H2O2 + Cl- H2O + O-Cl-
Myeloperoxidase
a)
b)
ROS
Figure 9.8 Generation of reactive oxygen species (ROS) in granulocytes. a: Sum-mary of the enzymatic reactions involved. b: Release of enzymes into a phagosomecontaining an ingested bacterium generates ROS and kills the bacterium.
9.5.3 Scavenging of reactive oxygen species
Where not needed for immune defense, reactive oxygen species are harmfulrather than useful. Nevertheless, some ROS always form as byproducts of res-piration, or even of oxygen transport in erythrocytes, since binding to hemeoffers oxygen an opportunity to steal an electron. This toxicity is controlledby the presence of glutathione (Figure 9.9a). An important step in the detox-ification of reactive oxygen is the reduction of hydrogen peroxide (H2O2) towater by glutathione peroxidase (Figure 9.9b). Glutathione is then regeneratedby glutathione reductase at the expense of NADPH.
9.6 Glucose-6-phosphate dehydrogenase deficiency
Glucose-6-phosphate dehydrogenase is an essential enzyme, and complete lackof it is not compatible with life. However, there are hereditary mutations
116 9 The hexose monophosphate shunt
O
O
SH
O
NH
NH
O
NH2
O
O
O
O
S
O
NH
NH
O
NH2
O
O
O
O
S
O
NH
NH
O
NH2
O
O
a)
b)
Glutathione peroxidase
H2O2
2 H2O
2 G-SH
G-SS-G NADPH+H+
NADP+
Glutathione reductase
c)
O2
H2O22 GSH
GSSG
2 GSH
GSSG
NH
N NH
O
O
CH3
NH
N NH2
OH
O
CH3
Glutathione peroxidase
spontaneous spontaneous
Figure 9.9 The role of glutathione in scavenging of reactive oxygen species, andthe mechanism of glutathione depletion in favism. a: Glutathione in its reduced andoxidized states. b: Glutathione peroxidase oxidizes glutathione and reduces hydrogenperoxide to water. Reduced glutathione is regenerated using NADPH by glutathionereductase. c: Isouramil is one of several similar compounds found in broad beans.It cycles between reduced and oxidized states; each cycle oxidizes four molecules ofglutathione.
9.6 Glucose-6-phosphate dehydrogenase deficiency 117
characterized by reduced enzyme activity. The defect becomes manifest mostlyin erythrocytes. This is related to the fact that erythrocytes have no proteinsynthesis, which has two consequences:
1. All protein molecules, including enzymes, have to last for the entirelifetime of the cell, which is normally 120 days. In contrast, in nucleated cellsthe lifetime of an enzyme molecule is typically in the range of hours to a fewdays. Reduced intrinsic stability of the enzyme may be masked by fast turnoverin nucleated cells but become evident in red cells.
2. In nucleated cells, there typically is a mechanism that adjusts enzymeexpression to metabolic needs; reduced specific enzyme activity may thereforebe partially compensated by increased expression. No such mechanism existsin red cells.
If the enzyme defect becomes manifest, the reduced throughput of the hex-ose monophosphate shunt will limit the regeneration of glutathione. Therefore,oxidative stress will result in glutathione depletion, which may lead to cell dam-age. In most people, the glucose-6-phosphate dehydrogenase defect does notshow up until they ingest some drug or food constituent that places increasedoxidative stress on the red cells. The classical trigger is the broad bean, viciafava, and the condition is therefore known as favism. The broad bean containsseveral pyrimidine bases that undergo redox reactions with both oxygen andglutathione. This leads to a catalytic cycle, in which each molecule of base oxi-dizes many molecules of glutathione, which in turn is reduced at the expenseof NADPH. This is illustrated for one of these bases, isouramil, in Figure 9.9c.
Ingestion of broad beans results in consumption of NADPH in both healthyindividuals and those afflicted by glucose-6-phosphate dehydrogenase defi-ciency. However, while healthy individuals can compensate this, NADPH be-comes depleted in those with the enzyme defect. The red cells will then sufferdestruction (= hemolysis) at the hands of unscavenged reactive oxygen species.
Hemolytic crises can also be caused by certain drugs, including anti-infectiousagents such as sulfonamides and metabolites of the anti-malarial drug pri-maquine, which can undergo cyclical oxidation and reduction just like isouramil.To what extent this effect is involved in the therapeutic effect of primaquine inmalaria is unclear. So, it is very important to watch out for glucose-6-phosphatedehydrogenase deficiency in patients to be treated with one of these drugs.
Like sickle cell anemia and other hemoglobinopathias, glucose-6-phosphatedehydrogenase deficiency is particularly common in areas with widespreadoccurrence of malaria. Like sickle cell anemia, it supposedly affords some pro-tection against malaria; the biochemical mechanism of this protection remainsunsettled.
Chapter 10
Triacylglycerol metabolism
Various types of lipids occur in the human body: (1) Triacylglycerol, (2) choles-terol, and (3) polar lipids, which include phospholipids, glycolipids and sphin-golipids.
While polar lipids and cholesterol are found in the cell membranes of ev-ery cell, triacylglycerol is essentially confined to fat tissue, which stores andreleases it, and to the cells in the intestine and the liver that synthesize anddegrade it.1 Yet, triacylglycerol is the most abundant lipid species, and the onlyone with an important role in energy metabolism. We will therefore here focuson triacylglycerol. Cholesterol, which is not important in energy metabolism,will be covered in a separate chapter as well because of its medical importance.
Triacylglycerol occurs in human metabolism in two roles: (1) As a foodstuff.A significant fraction of our caloric intake is triacylglycerol. (2) As an endoge-nous store of metabolic energy. This storage can be replenished from dietaryfat, or by endogenous synthesis of fat from carbohydrates or proteins.
The amount of energy per gram of tissue is far higher in fat than in anyother tissue, for two reasons: (1) One gram of triacylglycerol itself containsroughly twice as many calories as one gram of carbohydrates or protein. This isbecause triacylglycerol contains much less oxygen than carbohydrates, in whichoxygen contributes half the mass but essentially no metabolic energy. Similarly,
1There is also some intracellular storage of triacylglycerol, for example in the muscle cells,but it is quantitatively insignificant relative to adipose tissue.
118
Triacylglycerol metabolism 119
O O O
O O O
CH2
CH
CH2
OHOHOH
Glycerol
OH
CH3
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
O
Palmitic acid (hexadecanoic acid)
a)
b)
fatty acids
glycerol
acetyl-CoA
pyruvateglucose
Triacyl-glycerol
CO2 + H2O
ADP ATP
ketone bodies
14 7
5
2
36
Figure 10.1 Overview of triacylglycerol metabolism. a: Structure of tripalmitin, anexample species of triacylglycerol. Three molecules of fatty acid—here: palmitic acid—are esterified to the three hydroxyl groups of glycerol. b: Pathways: Triacylglycerolbreakdown (1) and synthesis (2), β-oxidation (3), fatty acid synthesis (4), ketone bodysynthesis (5) and utilization (6), TCA and respiratory chain (7). The dashed line repre-sents the “Rubicon” between carbohydrates and fat: Once pyruvate is decarboxylated,the remaining carbon cannot be turned back to glucose anymore.
120 10 Triacylglycerol metabolism
protein contains oxygen, nitrogen and sulfur and is similar in energy contentper dry weight to carbohydrates. (2) Triacylglycerol in fat cells coalesces todroplets that are entirely free of water. In contrast, protein and carbohydrates,including glycogen, always remain hydrated.
Because of this efficiency of storage, it makes sense that most of the excessglucose or protein is converted to fat, while only a limited fraction is stored asglycogen. There is, however, one limitation to the usefulness of triacylglycerol:Once carbohydrate or protein carbon has been converted to fat, it can go backand forth between fat, fatty acids, acetyl-CoA, and ketone bodies, but it is nolonger available for the regeneration of glucose (Figure 10.1b). Therefore, whenstarving, we will always have to degrade some protein along with fat to keepup a minimum supply of glucose, and therefore starving people from day zerowill not only deplete their fat stores but also their muscle tissue.
10.1 Utilization of dietary triacylglycerol
10.1.1 Solubilization and digestion
Digestion of triacylglycerol requires two components: (1) Bile acids. Theseact as detergents and solubilize the chunks or droplets of ingested fat intosmall micelles and in this way render the triacylglycerol accessible to enzy-matic cleavage. One of the major bile acids, cholic acid, is shown in Figure 11.1.(2) Pancreatic lipase. This esterase hydrolyses triacylglycerol to 2-monoacyl-glycerol and two molecules of free fatty acids.
The enzyme can act upon the triacylglycerol molecules only at the surface ofthe micelles. Dispersal of the lipid particles by bile acids is therefore necessaryfor hydrolysis. The fatty acids released by lipase act as detergents themselvesand will aid in the solubilization of remaining fat (Figure 10.2).2
After solubilization and hydrolysis by the lipase enzymes, the monoacyl-glycerol and the free fatty acids are taken up by the intestinal epithelial cells.If either bile acids or pancreas lipase are lacking—due to malfunctions of liveror pancreas, or to obstruction of the respective secretory ducts—fat malab-sorption will occur, and fat will appear in the stool. In addition, fat-solublevitamins (A, D, E, K) will be carried down the drain along with the fat, andvitamin deficiencies may ensue.
10.1.2 Formation of chylomicrons
After the products of triacylglycerol digestion—monoacylglycerol and fattyacids—have been taken in the small intestine, the next thing to happen to themis somewhat surprising: They are directly converted back to triacylglycerolwhile still inside the intestinal epithelial cells. The newly formed fat is then
2Curd soap is prepared simply by alkaline hydrolysis of fat; it consists of the sodium salts ofthe fatty acids released.
10.1 Utilization of dietary triacylglycerol 121
c)
total detergent concentration
monomer
micellar
CMC
a)
Detergent micelle
Monomeric detergent
Fat
Mixed micelle (solubilized fat)
b)
O
OH O OHO O
Figure 10.2 Fat solubilization by detergents. a: Monomeric detergent moleculespenetrate the surface of a fat droplet and break it up into mixed micelles. b: Cleavageof triacylglycerol by lipase yields 2-monoacylglycerol and free fatty acids, which actas detergents and assist in the solubilization of remaining fat. c: Detergents mayexist in micellar and monomeric form. Below its critical micellar concentration (CMC),a detergent dissolves completely as monomers; any excess above the CMC will formmicelles (cf. panel a, left). Bile acids have very high CMC values. They therefore havehigh concentrations of available monomers and solubilize fat rapidly.
122 10 Triacylglycerol metabolism
combined with protein molecules called apolipoproteins into lipoprotein par-ticles, such that the proteins form a hydrophilic shell around the lipid core(Figure 10.3a). Some phospholipids are included as well and complete thehydrophilic shell.
Apolipoproteins and lipoproteins occur in various subtypes (section 11.1).The specific lipoprotein particles formed at this stage, the chylomicrons, are thelargest ones among all lipoproteins, with a molecular mass of up to 1010 Dalton,a diameter up to 1 µm, and approximately 107 molecules of triacylglycerol. Thechylomicrons are released from the intestinal cells at the basolateral side. Sincethey are so large, they cannot diffuse acrosse the capillary walls to reach theblood stream—which small molecules can—but are instead drained into thelymphatics. All lymph fluid is ultimately collected in the thoracic duct, whichin turn discharges into the main circulation, bypassing the liver. Therefore,triacylglycerol is an exception from the general rule that all substances takenup in the intestine are first passed through the liver.
10.1.3 Utilization of chylomicrons
The triacylglycerol contained in the chylomicrons can be utilized in variousways: (1) It can be stored in fat cells, or (2) it can be utilized directly for thepurpose of ATP production by muscle cells and other tissues.
In both cases, the triacylglycerol in the chylomicrons again needs to getacross the barrier of the vascular endothelium. This is accomplished withthe help of lipoprotein lipase, an enzyme found at the endothelial surface. Itbinds the chylomicrons and cleaves the triacylglycerol again to fatty acids andglycerol. These are small molecules and can diffuse across the endothelialbarrier to reach the cells in the surrounding tissue. In fat cells, the fatty acidsare combined with glycerol yet again for storage (Figure 10.3b). Alternatively, inother cells, they can be directly degraded to acetyl-CoA, which is then consumedin the TCA and the respiratory chain. The remnants of chylomicrons, depletedof most of the triacylglycerol, are captured by the liver,3 phagocytosed, anddegraded.
Why is the transport of triacylglycerol so complicated? It would seem easierto do away with all these splitting and rejoining steps and just pour the fattyacids into the blood. One reason is that fatty acids are toxic. As mentionedbefore, they are detergents – and detergents solubilize cell membranes, that isthey cause cell destruction.4
3Sooner or later, each particle in the blood will pay the liver a visit, even if it was initiallybypassed.
4Interestingly, the surfaces of the intestinal cells withstand high concentrations of bile andfatty acids! This phenomenon has not yet received appropriate scientific attention.
10.1 Utilization of dietary triacylglycerol 123
b)
a)
Endothelial cell
Fat cell
Fatty acids,glycerol
Lipoprotein lipase
Chylomicron
Triacylglycerol
to Liver
Fatty acids, 2-MAG
Fatty acids, 2-MAG
ATP
ADP
Triacylglycerol
Apolipoproteins
Chylomicrons
Chylomicrons
intestinal epithelium
lymphatics
Protein
Triacyl-glycerol
Figure 10.3 Intestinal uptake and post-processing of dietary fat. a: Left: Fatty acidsand monoacylglycerol are taken up by the intestinal cells, converted back to triacyl-glycerol, and together with apolipoproteins packaged into chylomicrons. Right: Chy-lomicrons (and other lipoproteins) are fat droplets surrounded by a protein shell. b:Lipoprotein lipase binds chylomicrons and extracts and cleaves triacylglycerol. Thecleavage products are stored in fat cells (after being converted to triacylglycerol yetagain, as shown) or utilized by oxidative degradation.
124 10 Triacylglycerol metabolism
10.2 Utilization of fatty acids: β-Oxidation
As briefly mentioned before, the utilization of fatty acids occurs by way ofconversion to acetyl-CoA, which is accomplished in β-oxidation. This pathwayruns in the mitochondria, so the first task after cellular uptake of the fatty acidmolecule is to get it into the mitochondrion.
10.2.1 Mitochondrial transport of fatty acids
Fatty acids are initially activated to fatty acyl-CoA in the cytosol. This is alsothe form in which they enter degradation by β-oxidation. However, duringtransport, the CoA-moiety is transiently replaced by carnitine. The process isoutlined in Figure 10.4.
One unusual aspect of this transport process is that the energy of the thi-oester bond in acyl-CoA seems to be sufficiently well preserved in the esterbond of acylcarnitine to allow the exchange reaction to be reversed inside themitochondrion. Carboxyl esters bonds don’t usually have a sufficiently highenergy content for that. I once read a research paper on that topic, but it wentstraight over my head. I will therefore have to owe you the explanation whycarnitine is special in this respect. On the bright side, you won’t have to knowit in the exam.
10.2.2 Reactions in β-oxidation
The term β-oxidation refers to the greek lettering of the carbons in organicmolecules: The α carbon is right next to a functional group, and the β carbonis the second one (Figure 10.5a, top). It is at the second carbon from thioester,then, where the action is in β-oxidation. Figure 10.5a gives a summary of thereactions:
1. The fatty acyl-CoA molecule is first dehydrogenated between the α andthe β carbon atoms by acyl-CoA dehydrogenase. FAD accepts the hydrogenabstracted and is reduced to FADH2. This yields 2-trans-enoyl-CoA.
2. The trans double bond just created is hydrated—that is, water is addedacross it—by enoyl-CoA hydratase, which yields hydroxyacyl-CoA. The α carbonis now once more fully reduced.
3. The β-hydoxyl group is converted to a keto group by hydroxyacyl-CoAdehydrogenase. NAD+ accepts the hydrogen. The product is β-ketoacyl-CoA.
4. Thiolase introduces a new molecule of coenzyme A to cleave the β-ketoacyl-CoA, to release acetyl-CoA and a new, shortened acyl-CoA that entersthe next cycle of β-oxidation.
The process is repeated until the fatty acid is completely broken down. Inthe case of even-numbered acyl chains, this will yield acetyl-CoA only, whereasodd-numbered acyl chains will yield one molecule of propionyl-CoA in the finalstep (Figure 10.5b). There is a special pathway to take care of the propionyl-CoA,which is surprisingly complicated (see below).
10.2 Utilization of fatty acids: β-Oxidation 125
OM IM
CAT I CAT II
Acyl-CoA Acyl-CoA
Acyl-carnitine
CoA
Acyl-carnitine
Acyl-CoA
Carnitine CoAFatty acid
CoA
ATP
ADP
ON
+
CH2
CH
CH2
O
CH3
CH3
CH3
COOH
S
O
CoA
Acyl-carnitine
Acyl-CoA
a)
b)
Carnitine
Figure 10.4 Carnitine-mediated transport of fatty acids to the mitochondrial matrix.a: Structures of acylcarnitine and of acyl-CoA. b: Fatty acids are activated in the cytosolto acyl-CoA by acyl thiokinase. After transport across the outer mitochondrial mem-brane, the acyl group is transferred to carnitine by carnitine acyltransferase. Exchangefor free carnitine transports the acylcarnitine molecule into the mitochondrial matrix,where carnitine is replaced again by coenzyme A by a second acyl transferase.
10.2.3 Reaction mechanisms in β-oxidation
I hope that you noticed the similarities of the enzyme reactions discussedabove to some others we have seen before. At least in the first three steps,the similarity to reactions in the citric acid cycle—succinate dehydrogenase,fumarase, and malate dehydrogenase—are quite straightforward. Also noticethe consistent use of redox coenzymes (NAD+ and FAD) in the two pathways:1. Whereever a CH−OH bond is dehydrogenated, NAD+ or NADP+ is employed
126 10 Triacylglycerol metabolism
a)
b)
S
O
CoA
O
α
β
γ ω
S
O
CoA
S
O
CoA
OH
S
O
CoA
SCoA
O
SCoA
OCH3S
O
CoAβ
1
2
3
4
1
FAD
FADH2
NAD+
NADH++H+
CoA-SH
SCoA
O
SCoA
O
CS
O
CoACH3
CH3S
O
CoA
Acetyl-CoA
Propionyl-CoA
Figure 10.5 Reactions in β-oxidation. a: Enzymes: 1, acyl-CoA dehydrogenase; 2,enoyl-CoA hydratase; 3, hydroxyacyl-CoA dehydrogenase; 4, thiolase. The thiolase reac-tion yields acetyl-CoA and a new, shortened molecule of fatty acyl-CoA that undergoesa new round of β-oxidation. b: After completion of β-oxidation, even-numbered acylchains will yield acetyl-CoA only, whereas the last unit released from an odd-numberedacyl chain will be propionyl-CoA.
as the cosubstrate.5 2. Where dehydrogenation occurs across a CH−CH bond,
5This also holds in other pathways and for CH−NH bonds. The only exception I’m aware of isthe dehydrogenation of glycerolphosphate in the glycerolphosphate shuttle.
10.2 Utilization of fatty acids: β-Oxidation 127
S
O
CoAO
CoA S
S
O
CoA
O
Enzyme S
H+
B
B
H+
Enzyme S
BO
Enzyme S
SCoA
Figure 10.6 Mechanism of the thiolase reaction. See text for details.
FAD is employed.
As to the thiolase reaction, we have not seen a completely analogous one.However, if we look at the individual steps of this reaction, we can still recog-nize some familiar features (Figure 10.6):
1. The nucleophilic attack of a cysteine sulfur in the active site on a carbonylgroup in the substrate yields a covalent intermediate. This also happens withglyceraldehyde-3-phosphate dehydrogenase.
2. Acid-base catalysis breaks a carbon-carbon bond adjacent to the carbonylbond. Note that the thiolase reaction is reversible. Making of a carbon-carbonbond adjacent to the carbonyl group by acid-base catalysis then occurs in thereverse reaction, and we have seen that before with citrate synthase and malatesynthase.
3. The creation of one thioester at the expense of another occurs in thesecond step of the pyruvate dehydrogenase reaction.
10.2.4 Utilization of propionyl-CoA
Odd-numbered fatty acids yield one molecule of propionyl-CoA propionyl-CoAutilization as the final degradation product. This metabolite has an elabo-rate degradative pathway (Figure 10.7):
1. Initially, propionyl-CoA is converted to S-methylmalonyl-CoA by propi-onyl-CoA carboxylase. This reaction uses CO2 and ATP; biotin functions asa coenzyme. The reaction mechanism is the same as in the case of pyruvatecarboxylase (section 7.1).
2. S-methylmalonyl-CoA is converted to its enantiomer R-methylmalonyl-CoA by methylmalonyl-CoA racemase.
128 10 Triacylglycerol metabolism
CH2S
O
CoACH3
CH2S
O
CoACH2
COOH
CO2
C S
O
CoACH3
COOH
S
O
CoACCH3
COOH
(S)-Methylmalonyl-CoA (R)-Methylmalonyl-CoA
ATP
ADP
Propionyl-CoA Succinyl-CoA
1
2
3
Figure 10.7 Utilization of propinyl-CoA. Enzymes: 1, propionyl-CoA carboxylase; 2,Methylmalonyl-CoA racemase; 3, Methylmalonyl-CoA mutase.
3. Finally, one carboxyl group is transplanted within the molecule by methyl-malonyl-CoA mutase to yield succinyl-CoA. This is a frighteningly complex re-action; we will skip the gory details and just state that it requires Vitamin B12(cobalamine) as a coenzyme.
Note that succinyl-CoA is a citric acid cycle intermediate. It therefore canbe converted to glucose, so that propionyl-CoA is an exception to the rule thatcarbon from fatty acids cannot be used for gluconeogenesis. It seems, however,that fatty acids with odd numbers of carbons account only for a small fractionof all fatty acids, so that this exception is not quantitatively very important.
10.2.5 Energy balance of β-oxidation
The energy-rich metabolites accumulated in β-oxidation—NADH, FADH2, andacetyl-CoA—are degraded in the TCA and the respiratory chain as describedpreviously. The exact amount of ATP derived—with the usual assumptions of3 ATP per NADH and 2 per FADH2—strikes me as something suitable for anexam question.
10.3 Triacylglycerol utilization
When triacylglycerol is mobilized from the fat tissue, it is cleaved by an enzymenamed hormone-sensitive lipase. This lipase is stimulated by glucagon andepinephrin, the antagonist hormones of insulin. The cleavage products, glyceroland free fatty acids, are released into the plasma, where the fatty acids bindto albumin for transport. The fatty acids can be utilized in two different ways(Figure 10.8):
10.3 Triacylglycerol utilization 129
glycerol+ FA
TAG
acetyl-CoA
gluconeogenesis glycolysis
ketogenesis
glucose
acetyl-CoA
ketone bodies
FA
Figure 10.8 Overview of organ relationships in fat utilization. Triacylglycerol iscleaved in fat tissue to fatty acids and glycerol. Fatty acids are utilized by β-oxidationin heart and skeletal muscle, or first converted to ketone bodies in the liver. Ketonebodies can be consumed by many organs including the brain. Glycerol is utilized forgluconeogenesis.
1. Directly, that is they are taken up by the energy-requiring target tissueand degraded by β-oxidation. You will recall that β-oxidation is preceded bythe translocation to the mitochondrion, which requires carnitine. About 95% ofall carnitine is found in skeletal muscle,6 so that it appears that muscle tissueis the major client for direct utilization. The heart muscle cells also consumefatty acids by β-oxidation.
2. Indirectly, after initial conversion to ketone bodies in the liver (Figure10.9). This term comprises two small organic acids, acetoacetate and β-hydrox-ybutyrate. The brain, which in the well-fed state only utilizes glucose for ATPregeneration, is able to adapt to ketone bodies during starvation. The brainis responsible for approximately 20% of our total energy consumption at rest,and therefore is a major consumer of ketone bodies. Other major consumersare heart and skeletal muscle.
The second cleavage product of fat, glycerol, is picked up by the liver aswell. It is phosphorylated by glycerol kinase to glycerolphosphate. As in theglycerolphosphate shuttle (section 6.5), this metabolite is dehydrogenated todihydroxyacetone phosphate and can then be utilized for gluconeogenesis.Glycerol released from fat therefore contributes to keeping up a minimumsupply of glucose in times of starvation.
6Carnis is Latin for meat – as in chili con carne.
130 10 Triacylglycerol metabolism
10.3.1 Brown fat tissue
You know from experience that fat tissue is mostly white, which is due tothe fact that typical fat cells contain little else than triacylglycerol droplets.Intensely colored tissues (other than the pigment cells of the skin) are richin and owe their color to heme and / or cytochromes. This is the case withbrown fat tissue. The color of this special type of fat tissue derives fromthe abundant presence of mitochondria, which in turn contain cytochromes.They also contain a high amount of an uncoupling protein (section 6.1) calledthermogenin.
In contrast to white fat cells, brown fat cells do not release fatty acidsinto the circulation but instead perform β-oxidation themselves. The derivedhydrogen is oxidized in the respiratory chain. However, the resulting proton-motive force is not used for ATP synthesis but instead simply dissipated asheat – the purpose of brown fat is heat production. There is little brown fat inadult humans or most other adult mammals. However, there is a substantialamount in newborns, who because of their higher surface-to-volume ratio (andtheir helplessness) are more prone to hypothermia. Brown fat is also foundin hibernating animal species such as polar bears, which need it to reheatthemselves to operating temperature during arousal from hibernation.
10.4 Ketone bodies
Ketone bodies can be considered a water-soluble and non-toxic transport formfor the carbon mobilized from fatty acids. Ketone body synthesis occurs in themitochondria, that is in the same compartment as β-oxidation. It involves thefollowing reactions (Figure 10.10):
1. Formation of acetoacetyl-CoA from two molecules of acetyl-CoA by thi-olase (Figure 10.10a). This step is the reversal of the final step in β-oxidation.
2. Formation of hydroxymethylglutaryl-CoA (HMG-CoA) by HMG-CoA syn-thase (Figure 10.10b, 1).
3. Release of acetoacetate by HMG-CoA lyase (Figure 10.10b, 2);
4. Reduction of acetoacetate to β-hydroxybutyrate by β-hydroxybutyratedehydrogenase (Figure 10.10c).
Acetoacetate can form acetone (CH3−CO−CH3) through slow, spontaneousdecarboxylation. Acetone cannot be metabolized; it is volatile and is exhaled.A characteristic acetone smell can be detected in persons that utilize fat at ahigh rate.
One such situation is diabetic ketoacidosis. If insulin is lacking, fat cellscannot import glucose. They mistake this for a state of starvation and startbreaking down fat, which is then converted to ketone bodies. If you detect an
10.4 Ketone bodies 131
OO
OH
OHO
OH
a)
b)
FA + glycerol
AcetoacetateFA Acetyl-CoA
FAD FADH2
NAD+ NADH+H+
Acetoacetateβ-HB
TAG
β-HBNAD+
NADH+H+
Acetoacetyl-CoA
Acetyl-CoA
Succinyl-CoA
Succinate
TCA
Figure 10.9 Ketone body metabolism. a: Structures of acetoacetate and β- hydroxy-butyrate (β-HB). b: Overview of metabolic pathways. Triacylglycerol is cleaved to fattyacids and glycerol in the fat tissue. Fatty acids undergo β-oxidation in the liver. Acetyl-CoA is converted to acetoacetate, which is released into the blood. Half of the NADHgenerated in β-oxidation is used to convert acetoacetate to β- hydroxybutyrate. In thebrain and other tissues, the two substrates are converted back again to acetyl-CoA andutilized in the citric acid cycle.
acetone smell in an unconscious person, this is highly suspicious of diabeticcoma (see section 14.4).
Another condition with accelerated breakdown of fat is plain ol’ flu andother states of fever. Metabolism is accelerated by fever; at the same time,patients often have little or no appetite. Fat is broken down, and again anacetone smell may develop. This by itself does not indicate a serious condition.
Utilization of ketone bodies in the brain, muscle and other tissues is straight-forward (Figure 10.9): (1) β-Hydroxybutyrate is dehydrogenated to acetoacetate,(2) acetoacetate steals coenzyme-A from succinyl-CoA to become acetoacetyl-CoA, (3) thiolase cleaves acetoacetyl-CoA to acetyl-CoA.
132 10 Triacylglycerol metabolism
CH3S
O
CoA
O
CH2 S
O
CoAH
Enzyme-B
OH2
CoA
CH2 OH
O
CH3S
O
CoA
OH
Acetyl-CoA
CH3OH
O O
CH2 S
O
CoA
CH3S
O
CoA
O
Enzyme B+
H
1
1
2
CH3
C
CH2
COH
O
OH
H
CH3
C
CH2
C
O
OH
ONAD+NADH+H+
a)
b)
c)
CH3 S
O
CoA
O
H+
B
CH3
O
CoA S
SEnzyme
Enzyme S CH3
O
B
CH2 S
O
CoAH
CoA-SH
Figure 10.10 Reactions in ketone body synthesis. a: Formation of acetoacetyl-CoAby thiolase. This reaction is the reversal of the cleavage occurring in β-oxidation.b: Synthesis of hydroxymethylglutaryl-CoA (HMG-CoA) by HMG-CoA synthase (1) andrelease of acetoacetate by HMG-CoA lyase(2). c: Reduction of acetoacetate to D-β-hydroxybutyrate by β-hydroxybutyrate dehydrogenase.
10.5 Fatty acid synthesis 133
CH3S
O
CoA CS
O
CoAC
OH
O
ATP ADP
CO2
Figure 10.11 The reaction catalyzed by Acetyl-CoA carboxylase.
10.5 Fatty acid synthesis
Fatty acids can be synthesized from acetyl-CoA. This is the major way of uti-lizing excess dietary carbohydrates. Fatty acid synthesis occurs mainly in thefat tissue and the liver. Synthesis and degradation of fatty acids are similar inthat they involve a cycle of reactions that changes the length of the substrateby two carbon atoms at a time. However, they differ from each other in severalaspects:
1. Synthesis runs in the cytosol, whereas β-oxidation occurs in the mito-chondria. This makes it easier to control the two pathways separately.
2. Synthesis uses NADPH rather than NADH and FADH2 to reduce the C=Oand C=C double bonds. Use of NADPH drives both reactions toward reduction.
3. The C2 subunits that are sequentially added are derived from malonyl-CoA rather than from acetyl-CoA directly. Again, this thermodynamically fa-vours synthesis over degradation.
Points 2 and 3 both conspire to make fatty acid synthesis irreversible. Incontrast, β-oxidation is reversible, and reversed β-oxidation is actually used bymitochondria to synthesize their own fatty acids.
The bulk of the work in fatty acid synthesis is accomplished by fatty acidsynthase, which is quite an amazing molecule: It combines eight distinct cat-alytic activities on a single polypeptide chain. Its product is palmitic acid(hexadecanoic acid; sixteen carbons, fully saturated). As stated before, fattyacids vary in their chain lengths and degree of bond saturation. Chain elonga-tion and desaturation is accomplished by separate enzymes, called elongasesand desaturases. We just note these here but will not consider them in detail.
10.5.1 Reactions in fatty acid synthesis
The only reaction in fatty acid synthesis that is not carried out by fatty acidsynthase is the first one, which is catalyzed by acetyl-CoA carboxylase. Thisreaction is depicted in Figure 10.11. I hope that, by now, you recognize thepattern: CO2 is fixed, and ATP is expended – another biotin-dependent reaction,operating in the same way as discussed before for pyruvate carboxylase andpropionyl-CoA carboxylase.
All further reactions are catalyzed by fatty acid synthase. In animals, this isa large molecule with all active sites on a single polypeptide chain. Two of thesepolypeptides form an active dimer. The overall structure shown in Figure 10.12
134 10 Triacylglycerol metabolism
has been determined using electron microscopy and image processing. Thecrystal structure has recently been determined as well (Science 321:1315-22,2008).
The growing fatty acyl chain remains covalently attached to the enzymethroughout the entire synthesis process. Attachment is through a phospho-pantetheine group, which provides a flexible tether, enabling the acyl chain totravel and visit the various active sites on the enzyme in turn (Figure 10.12c).This is reminiscent of the lipoamide moiety in pyruvate dehydrogenase andof biotin-dependent carboxylases.7 The phosphopantetheine tether group isactually not new to us – it also occurs in coenzyme A (Figure 10.12b).
In Figure 10.13, the structure of fatty acid synthase has been rather drasti-cally simplified; only the two thiol groups, which serve as points of covalentattachment of acyl-CoA and malonyl-CoA, are shown. Keep in mind, however,that they are close to each other only in two reactions (3 and 7 in Figure 10.13).For the other reactions, the tethered acyl residue swings back and forth to in-teract with the other active sites. The reactions catalyzed by fatty acid synthaseare as follows (see Figure fatFAsynthesis):
1. Transfer of an acetyl-group from acetyl-CoA to the active-site cysteine.Both in the substrate and the product, the malonyl group is bound as a thioester,so that this reaction is easy; a similar exchange is part of the thiolase reaction(Figure 10.10a).
2. Transfer of a malonyl group from malonyl-CoA onto the phosphopan-tetheine group of the FA synthase. Since the two carriers are so similar, this isagain a very straightforward reaction.
3. Condensation of the malonyl group to the end of the acetyl group. Apartfrom the concurrent decarboxylation (which facilitates the transient formationof a carbanion), this reaction resembles the reversal of the thiolase reaction inβ-oxidation.
4. Reduction of the keto group to a hydroxyl group,5. Elimination of water to create a C=C double bond and6. Reduction of the latter to a single bond. These steps are reversals of those
occuring in β-oxidation, except that NADPH is used as the redox coenzyme.7. Transfer of the extended acyl chain from the phosphopantheteine group
to the aforementioned active site cysteine. Note that this reaction is actuallyvery similar to the initial transfer of an acetyl group from acetyl-CoA – in bothcases, transfer is from a pantetheine group to the cysteine.
8. The phosphopantetheine site is now free to accept another malonylgroup, and the cycle repeats, with the acetoacetyl group now taking the placeof the acetyl group in the first cycle.
7In E. coli, the phosphopantetheine group is associated with a separate small protein, the acylcarrier protein (ACP). In mammalian fatty acyl synthesis, ‘ACP’ is not a separate protein but ispart of the synthase molecule itself.
10.5 Fatty acid synthesis 135
Cys-S-
a)
b)
c)
O
P
O
OO CH2
C C C NH
CH3
CH3 OH
H
O
CH2
CH2
C
O
NH
CH2
CH2
SSerEnzyme
O
P
O
OO CH2
C C C NH
CH3
CH3 OH
H
O
CH2
CH2
C
O
NH
CH2
CH2
SPAdenosine
O
O
S
O
**
Figure 10.12 Structure of fatty acid synthase. a: Electron microscopy. Left: Thewhite squares highlight individual molecules. (No, I can’t recognize them either.) Right:The three-dimensional structure, obtained by merging and averaging many images ofindividual molecules. The two subunits of the dimer are elongated and oriented inantiparallel fashion. The stars highlight the putative location of the active sites. From:Brink, Jacob et al. PNAS 99:138-143 (2002). b: Structure of the phosphopantetheinegroup (top) compared to coenzyme A. c: The phosphopantetheine group acts as a tetherfor the acyl group, which can therefore visit the various active sites of the synthase.The cysteine is located in one of the active sites (the one which has acyl transferaseactivity). Only one of the two enzyme subunits is shown.
136 10 Triacylglycerol metabolism
Cys S
O
Pant S CH3
OEnzyme
NADPH
NADP+
CO2
O
CH3Cys S
O
Pant S O
OEnzyme
Cys S
O
Pant S CH3
OHEnzyme
Cys S
O
Pant S CH3
Enzyme
Cys S
O
Pant S CH3
Enzyme
Cys S
O
CH3
Pant S
Enzyme
CH3 S
O
CoA
Pant S
Cys S
Enzyme
O
CH3Cys S
Pant S
Enzyme
CoA S
OH2
Malonyl-CoA
CoA S
Malonyl-CoA
CoA S
NADPH
NADP+
1
2
3
4
5
6
7
8
Figure 10.13 Reactions of fatty acid synthesis. “Pant” represents the pantetheinemoiety of fatty acyl synthase. See text for details.
10.5 Fatty acid synthesis 137
a)
Mitochondrial matrix
Cytosol
acetyl-CoA acetyl-CoA
citrate citrateATP
ADP
OA OA
malate
NADH+H+
NAD+
NADPH+H+
NADP+
CO2
pyruvatepyruvateCO2
ATP
ADP+Pi
1
3
2
4
5 CoA-SH
c) 2 acetyl-CoA
acetoacetyl-CoA
acetoacetate acetoacetateATP
ADP
CoA
CoA
2 acetyl-CoA
2 CoA
1
2
3
b) acetyl-CoA acetyl-CoA
citrate citrateATP
ADP
oxaloacetate
malate
NAD+NADH+H+
oxaloacetate
malate
NAD+ NADH+H+
5
22
1
Figure 10.14 Mechanisms for the transport of acetyl-CoA from the mitochondrion tothe cytosol for fatty acid synthesis. a: The textbook shuttle. Enzymes: 1, ATP-citratelyase; 2, malate dehydrogenase; 3, malic enzyme; 4, pyruvate carboxylase; 5, citratesynthase. b: A simpler yet more likely shuttle. Enzymes as in a). c: The acetoacetateshuttle. 1: reactions of ketogenesis; 2: acetoacetyl-CoA synthetase; 3: thiolase.
Finally, when the full length of 16 carbons has been reached, the palmitateresidue is hydrolyzed off the pantetheine residue instead of being transferredto the cysteine (not shown).
The dimeric nature of the protein raises two possibilities for its mechanism:(1) The two subunits operate independently, that is the acyl chain only visitsactive sites within the subunit to which it is bound. (2) The acyl chain undergoesreaction in one or more of the active sites on the other subunit.
Splitting the dimer into monomers leaves most of the enzyme activitiesintact, indicating that these reactions occur within the same subunit. However,the initial reaction in each cycle—the condensation reaction between the fullyreduced acyl chain and the next malonyl residue—only occurs in the dimer,which suggests that it may be catalyzed by the other subunit.
10.5.2 Export of mitochondrial acetyl-CoA
We have noted before that fatty acid synthesis occurs in the cytosol. Sinceacetyl-CoA is formed in the mitochondrion by pyruvate dehydrogenase, we face
138 10 Triacylglycerol metabolism
the problem of getting it out of the mitochondrion and into the cytosol.You may recall that there was a similar problem in β-oxidation. In that case,
the transport of acyl-CoA was accomplished by the carnitine carrier system(section 10.2.1). Since all reactions in the latter system are reversible and acetyl-CoA is also an acyl-CoA, we might assume that the carnitine carrier systemcould also help us out here. However, if it did transport both acetyl-CoA andlonger acyl-CoAs in both directions, it might set up a futile cycle of fatty acidsynthesis and degradation. Accordingly, acetyl-CoA is transported by othermeans. There seem to be several pathways that contribute to this transport(Figure 10.14):
1. The major mechanism uses the cytoplasmic enzyme ATP-citrate lyase.Citrate is exported by the tricarboxylate carrier system and cleaved; concomi-tantly, the acetate fragment is activated to acetyl-CoA using ATP. The problemthen arises how to dispose of the oxaloacetate. While the textbooks usuallyaccount for this as shown in Figure 10.14a, this mechanism is not only com-plicated but also unbalanced. Citrate can only be transported in exchangefor something. One possibility is malate, which suggests that the mechanismoutlined in Figure 10.14b is more reasonable.A fairly straightforward transport mechanism that is active at least in mice, butprobably also other mammals, is the export of acetoacetate, which is generatedin the mitochondria by the ketogenesis pathway (Figure 10.10). Acetoacetatecan be converted back to acetyl-CoA by cytosolic acetoacetyl-CoA synthetase,which uses ATP, and a thiolase (Figure 10.14c).
The acetoacetate shuttle has the advantage of the lowest ATP expenditure(one ATP per two molecules of acetyl-CoA translocated).
10.5.3 Supply of NADPH for fatty acid synthesis
While the hexose monophosphate shunt typically gets the most credit forNADPH production, it seems that fat cells and possibly also liver cells ob-tain most of their NADPH by an alternative mechanism that uses cytoplasmicNADP+-dependent isocitrate dehydrogenase (Figure 10.15). Isocitrate formedin the TCA can be translocated into the cytosol in exchange for α-ketoglutarate.The isocitrate dehydrogenase reaction produces NADPH in the cytosol and alsoreplaces the α-ketoglutarate. The mitochondrial isocitrate dehydrogenase stepis simply skipped, and the TCA resumes at the level of α-ketoglutarate.
Now, Watson, if you have been following closely, you will have observed thatthis will only satisfy half the need for NADPH, since we get only one mole ofisocitrate per mole of acetyl-CoA, but fatty acid synthesis requires two molesof NADPH per acetyl-CoA.
NADH/NADP+-transhydrogenase to the rescue: In Figure 6.14, it was de-scribed how transhydrogenase and NADP+-dependent isocitrate dehydrogenasecan conspire to regenerate isocitrate from α-ketoglutarate. Instead of being
10.6 Pharmacological inhibition of fatty acid synthase 139
Mitochondrial matrix Cytosol
citrate
isocitrate
α-ketoglutarate + CO2
NADP+
NADPH+H+
isocitrate
α-ketoglutarate
succinyl-CoA
NADP+
NADPH+H+
Figure 10.15 The role of cytosolic isocitrate dehydrogenase in the regeneration ofNADPH for fatty acid synthesis. See text for details.
consumed in a futile cycle as discussed then, this isocitrate can again be madeavailable for cytosolic NADPH regeneration.
10.6 Pharmacological inhibition of fatty acid synthase
Cerulenin is a fungal antibiotic that binds to and irreversibly inhibits fatty acidsynthase. Its structure resembles the β-keto-acyl intermediates (Figure 10.16a),and the active site to which cerulenin binds is the very same one that formsthe β-keto-acyl group by condensation. The cysteine in this site then reactswith the epoxide ring in the cerulenin and is alkylated. While epoxide drugsare not terribly popular because they are quite reactive and often toxic, thereis presently some interest in the development of fatty acid synthase inhibitors,modeled on the structure of cerulenin, for the treatment of obesity. While thisdoes not strike me as a compelling idea,8 a more interesting application is inthe treatment of cancer. Most non-cancerous cells do not express fatty acidsynthase, relying instead on fatty acids supplied by the liver and fat tissue.However, fatty acid synthase expression has been observed in cancer cells; thefatty acids they produce probably feed their synthesis of membrane glyco- andphospholipids, which they require for proliferation. Inhibition of fatty acidsynthase can induce apoptosis (programmed cell death) in tumor cells.
Figure 10.16b shows an experimental synthetic drug that is somewhat sim-ilar to polyphenols occurring in green tea and other natural sources. In mice
8I look forward to the discoveries of metabolic derailments these may cause in the face ofcontinued excess calory intake – what is going to become of the surplus acetyl-CoA if fatty acidsynthesis is inhibited: Ketone bodies? Cholesterol? Will glycolysis be backed up and diabetes beinduced? All of the above?
140 10 Triacylglycerol metabolism
compound 7
O
O
O
OH
OH
OH
O
OH
OH
OH
0
100
200
300
400
500
0 10 20 30 40
avg. tu
mor
volu
me (
mm
3)
days of treatment
untreated
compound 7
O
NH2
O
OH S Cys Enzyme
O
OH
O
O
NH2
O
O S Cys Enzyme
H+
a)
b)
Figure 10.16 Inhibitors of fatty acid synthase. a: Cerulenine. Top: Structure of aβ-keto-fatty acid (shown for comparison). Middle: Reaction of the active-site cysteineof fatty acid synthase with the epoxide moiety of cerulenin. Bottom: The resultinginactivated state. b: Structure of and tumor growth inhibition by an experimental fattyacid synthase inhibitor. The plot shows the growth of tumor grafts in mice, with andwithout treatment with the inhibitor. Redrawn from original data in Puig et al., Clin.Cancer Res. 15:7608-7615 (2009).
experiments, it substantially reduces the growth rate of transplanted cancer.While such experimental cancers are notorious for being easier to treat thanactual clinical cancer in humans, the results suggest that this line of researchis worth pursuing.
Chapter 11
Cholesterol metabolism
Contrary to popular belief, the biological role of cholesterol is not limited tobeing the bad guy. Instead, it has a number of essential physiological functions:
1. In each individual cell, it occurs as a major constituent of the plasmamembrane. Cholesterol controls physical properties of the membrane, whichare important for the function of membrane proteins such as receptors andtransporters. Depletion of membrane cholesterol cripples many functions ofcells.
2. Cholesterol is the precursor of bile acids, which are essential for fatdigestion.
3. Cholesterol is the precursor of all steroid hormones: Androgens, estro-gens, gestagens, glucocorticosteroids, mineralocorticoids, and calciferols. Someexamples are illustrated in Figure 11.1.
Cholesterol can both be obtained from the diet and be synthesized in humanmetabolism. However, we cannot degrade it; therefore, cholesterol has nosignificant role in energy metabolism. I’m going to cover it here anyway becauseits relationship to fat metabolism and its medical and physiological relevance.
141
142 11 Cholesterol metabolism
Table 11.1 Characteristics of the major lipoproteins. VLDL: Very low density lipopro-tein; LDL and HDL: Low and high density lipoproteins.
Chylomicrons VLDL LDL HDL
Density (g/ml) 0.95 0.95–1.0 1.02–1.06 1.06–1.12
Origin Small intestine Liver VLDL Liver
Role intransport
Transport ofdietary fat
Distribution oftriacylglycerolfrom liver
Distributionof cholesterolfrom liver
Transport ofexcesscholesterol toliver
Mostabundantconstituents
Triacylglycerol Triacylglycerol Cholesterol Protein,phospholipids,cholesterol
11.1 Uptake and transport of cholesterol
The structure of cholesterol (Figure 11.1, top left) has very little similarity withtriacylglycerol. However, they are both hydrophobic molecules, and thereforethe two lipids share the same major mechanism of transport in the bloodstream,which is by way of lipoproteins. This applies to both dietary and endogenouslysynthesized lipids.
Cholesterol that is taken up from the intestines initially becomes bound tochylomicrons. It may then be transferred from there to cells in the periphery—recall that chylomicrons bypass the liver—or be redistributed to other lipopro-tein species. Some cholesterol will remain with the chylomicron remnants thatare finally phagocytosed and degraded in the liver.
11.2 Distribution of cholesterol from the liver to the pe-riphery
The major site of cholesterol biosynthesis is—once more—the liver.1 The ma-jor lipoprotein species synthesized by the liver is known as very low densitylipoprotein (VLDL), which transports both cholesterol and triacylglycerol fromthe liver to the periphery. By extraction and exchange of triacylglycerol, thecomposition of circulating VLDL successively changes until it becomes low den-sity lipoprotein (LDL), which has a particularly high concentration of cholesterol.This lipoprotein species is removed from the circulation through endocytosis2
by cholesterol-requiring cells, for example in tissues that synthesize steroidhormones. Endocytosis requires a specific receptor, the LDL receptor. A genetic
1The liver has been dubbed the lab of the human body. I disagree: It is more productive, andit makes fewer mistakes than any lab I’ve seen so far.
2Note that uptake by cells other than vascular endothelia must be preceded by transcytosis—endocytosis followed by exocytosis on the opposite side—across the endothelial cells.
11.3 Transport of excess cholesterol to the liver 143
OH
CH3
OH
H H
Progesterone Estradiol
O
CH3
CH3
CCH3
O
H H
OH
CH3
CH3
CH3
CH3
CH3
H H
O
CH3
CH3
C O
CH2OH
H H
OH OH
Cortisol
O
CH3
OH
H H
CH3
Testosterone
CH3
CH3
OH
OH
O
O
O
Cholic acid
Cholesterol
Figure 11.1 Structures of cholesterol and of several molecules derived from it. Cor-tisol, a glucocorticoid, induces enzymes that increase blood glucose, such as thosenecessary for gluconeogenesis. Progesterone, estradiol, and testosterone are sexualhormones. All these hormones are steroid hormones. Cholic acid is a detergent and isimportant in intestinal fat solubilization.
deficiency of this receptor is associated with increased blood levels of LDL andincreased risk of heart attack and stroke (see section 11.9).
11.3 Transport of excess cholesterol to the liver
Excess cholesterol can be transported back from peripheral tissue to the liverby high density lipoprotein (HDL). Among all lipoproteins, this one has thehighest ratio of protein to lipid and therefore the highest density. A highlevel of HDL in the blood is associated with a decreased risk of cardiovasculardisease; therefore, HDL has been dubbed the ‘good cholesterol’ as opposed toLDL, the ‘bad cholesterol’. The liver can either recycle the cholesterol into new
144 11 Cholesterol metabolism
lipoproteins, or dispose of excess cholesterol through secretion into the bile(see later).
11.4 Esterification of cholesterol
Unmodified cholesterol has a preference for a superficial location in lipidphases (e.g., membranes), such that its polar hydroxyl group peeks out intothe aqueous phase. This −OH group can be esterified, so that it becomesshielded by a fatty acyl residue (Figure 11.2). The fatty acyl residue is pur-loined from phosphatidylcholine, one of the major phospholipids in mem-branes and lipoproteins. The enzyme catalyzing this exchange is known aslecithin:cholesterol acyltransferase (LCAT). Cholesterol esters partition into theinterior of lipoprotein particles, which greatly increases the particles’ transportcapacity for cholesterol.
11.5 Synthesis of cholesterol
Cholesterol biosynthesis occurs in animals but not in plants or fungi. The latterhave similar sterols for similar purposes, which however cannot be converted tocholesterol. Therefore, it is essential for animals—particularly for plant-feedingones, such as sheep, goat and vegetarians—to have a pathway for cholesterolbiosynthesis. This pathway is quite complex, involving some 30 enzymes, and Idon’t know them all myself. We will not look at all reactions in the biosynthesisindividually but only at the initial stages, up to lanosterol, which is the firststerol intermediate.
The first steps of the synthesis tie in with other metabolic pathways wehave seen before (Figure 11.3). Synthesis starts with Acetyl-CoA in the mito-chondrion, which is used to synthesize hydroxymethylglutaryl-CoA (HMG-CoA).These reactions also occur in ketogenesis (section 10.4). However, while theentire process of ketogenesis occurs in the mitochondrion, the formation ofHMG-CoA in sterol synthesis occurs in the cytosol; therefore, we have separateversions of HMG-CoA synthase in the two compartments.
All subsequent steps occur in the smooth endoplasmic reticulum. HMG-CoA reductase reduces HMG-CoA to mevalonate, which in turn is converted tovarious isoprene compounds. Several rounds of polymerization3 lead to thelinear hydrocarbon molecule squalene, which is then converted to lanosterol. Alengthy series of subsequent modifications leads to cholesterol.
The reactions of the synthetic pathway are shown in Figure 11.4. The reac-tion catalyzed by HMG-CoA reductase is the first committed step, which means
3I’m using the term loosely – the reaction mechanisms are different from typical chemicalpolymerization.
11.5 Synthesis of cholesterol 145
O
CH3
CH3
CH3
CH3
CH3
H
H
H
O
O
O
O
O
O
N+
CH3
CH3CH3
LCAT
a)
O
O
O
O
O
O
O
O
O
O
O
O
O
O
free cholesterol
cholesterol ester
b)
Figure 11.2 Esterification of cholesterol. a: One acyl chain of phosphatidylcholine(left) is transferred to cholesterol by LCAT (lecithin:cholesterol acyltransferase). b:Distribution of free cholesterol and of cholesterol esters in lipoprotein particles. Thefree cholesterol is confined to the surface; the esters distribute to the interior, whichincreases the transport capacity.
that from this point onwards the substrates have no other option than becom-ing a sterol.4 Therefore, HMG-CoA reductase is the main target of regulatory
4No quantitatively important one, at least. However, farnesyl-pyrophosphate is also used in
146 11 Cholesterol metabolism
Acetyl-CoA HMG-CoA Mevalonate (C6)
CO2
3 ATP
3 ADP
Activated C5Activated C10Activated C15
Squalene (linear C30) Lanosterol (C30) Cholesterol
HMG-CoA-reductase
Figure 11.3 Overview of cholesterol biosynthesis. The first committed step is thereduction of HMG-CoA to mevalonate, which then is activated by ATP for polymeriza-tion up to squalene. Squalene, the last linear metabolite, is converted to lanosterol, theMother of All Sterols.
mechanisms, which in turn is exploited in pharmacotherapy (see later).The product of HMG-CoA reductase, mevalonate, undergoes repeated phos-
phorylations. One of the phosphate groups is used together with decarboxyla-tion to introduce a C=C double bond; the other ones are used later in polymer-ization. The most interesting reaction is the cyclization of squalene, the finalpolymerization product. Following the introduction of an epoxy group, all butone of the double bonds are rearranged, resulting in the formation of the firststerol, lanosterol. The enzyme in question, squalene monooxygenase, belongsto the cytochrome P450 family.
Several more steps are required to convert lanosterol to cholesterol. Thelast intermediate, that is the immediate precursor of cholesterol, is 7-dehydro-cholesterol, which is also the precursor of cholecalciferol (see below).
11.6 The endoplasmic reticulum in steroid synthesis
As noted above, the site of all reactions in cholesterol biosynthesis except thefirst one is the smooth endoplasmic reticulum. This also applies to most fur-ther modifications that convert cholesterol to one or the other steroid hormone.The smooth ER hosts many different cytochrome P450 enzymes. One of theseis squalene monooxygenase; others are responsible for introducing the vari-ous hydroxyl and carbonyl groups into the sterol molecule in the syntheticpathways for the steroid hormones (Figure 11.1).
Many intermediates in the synthesis of cholesterol and of its derivativesare very hydrophobic and very poorly soluble in water; they therefore have toreside in an apolar environment. On the other hand, the enzymes, like almost
the post-translational modification of some membrane-associated proteins.
11.6 The endoplasmic reticulum in steroid synthesis 147
OH
+
C+
OH
HMG-CoA Mevalonate
OCCH2
CH2
CSCoA
O
OH
CH3 O
O C CH2
CH2
CH2
OH
OH
CH3O 3 ATP 3 ADP
2 NADPH+H+ 2 NADP+
CoA-SH
3-P-5-PP-mevalonate
CH2 CH2
CH2
O
CH3
P PCH3 CH
CH2
O
CH3
P P
CH3 CH
CH2
CH3
CH2
CH
CH2
O
CH3
P P
PCO2
Isopentenyl-PPDimethylallyl-PP
Geranyl-PP
P-P
CH3 CH
CH2
CH3
CH2
CH
CH2
CH3
CH2
CH
CH2
O
CH3
P PFarnesyl-PP
P-P
NADPH+H+
NADP+P-P
O C CH2
CH2
CH2
O
O
CH3O
P
P P
OH
squalene
lanosterol
NADPH+H+
NADP+
O2
H2O
1 2
3
4
5
6
7
8
9
Figure 11.4 Enzyme reactions in the synthesis of lanosterol, the sterol precursorof cholesterol. PP stands for pyrophosphate. Enzymes/reactions: 1, HMG-CoA reduc-tase; 2: Mevalonate kinase and phosphomevalonate kinase; 3: Phosphomevalonate-5-pyrophosphate decarboxylase; 4: Isopentenyl-pyrophosphate isomerase; 5: Geranyl-pyrophosphate synthase; 6: Farnesyl-pyrophosphate synthase; 7: Squalene synthase; 8:Squalene monooxygenase. 9: Reaction intermediates of the squalene monooxygenasereaction.
148 11 Cholesterol metabolism
Smooth endoplasmic reticulum
Mitochondria
Rough endoplasmic reticulum
Figure 11.5 Electron microscopy of the endoplasmic reticulum in steroid-hormonesynthesising cell. The organelle is delineated by highly convoluted membranes,wherein the reactions of sterol modifications proceed. Similarly, in many reac-tions in cholesterol synthesis, the hydrophobic substrates are located within theapolar membrane core. Reproduced, with permission from Roger Wagner, fromhttp://www.udel.edu/Biology/Wags/histopage/histopage.htm.
all proteins, are at least partially polar and cannot immerse completely insidethe same apolar environment. The solution to this problem is to perform thereactions at the interface of polar and apolar environments, that is at membranesurfaces. This means that the membrane of the smooth ER is not just there todelimit a separate compartment, but itself is a reaction compartment.
The membrane of the smooth endoplasmic reticulum is extensively bulgedand invaginated, which gives it a very large surface area. The throughput ofcholesterol synthesis should be proportional to this surface area. Accordingly,the development of the smooth ER is particularly prominent in cells that per-form sterol or steroid hormone synthesis (Figure 11.5).
11.7 7-Dehydrocholesterol and vitamin D3
An interesting branch of the cholesterol synthesis pathway leads to the synthe-sis of vitamin D3 (calciferol). Since it vitamin D3 can by synthesized in humanmetabolism, it is not strictly a vitamin. The compound is synthesized from7-dehydrocholesterol, which is the immediate precursor of cholesterol. Theconversion is a photochemical reaction: It requires the absorption of a UV pho-ton and therefore can only occur at the body surface, that is in the skin. Two
11.8 Regulation of cholesterol synthesis 149
successive enzymatic hydroxylations, which do not require any more UV light,then convert calciferol to 1,25-dihydroxycalciferol. This molecule is essentialin the uptake of calcium and phosphate from the gut.
A lack of 1,25-dihydroxycalciferol causes ricketts, a disease characterized bybone deformations.5 The importance of 1,25-dihydroxycalciferol is responsiblefor the variation of human skin colors. While dark pigment protects the skinfrom damage by UV irradiation, the UV photons gobbled up by the pigment arenot available for the synthesis of calciferol. When Homo sapiens left Africa forless sunny climates, the shortage of sunshine created a selective pressure forlighter skin, which increases the availability of photons for calciferol synthesis.
With the availability of calciferol as a food supplement, light skin color doesno longer seem to have any biological advantages – only the disadvantage ofhigher rates of skin cancer. However, it is probably a good idea for dark-skinnedpeople living in northern climates to be particularly conscientious about givingcalciferol (vitamin D) prophylaxis to their children.
11.8 Regulation of cholesterol synthesis
The biosynthesis of cholesterol, like that of many other metabolites, is regu-lated both by allosteric control of enzyme activity and by enzyme induction.The committed step is the reduction of HMG-CoA to mevalonate, and thereforeit makes sense that HMG-CoA reductase is the primary target of regulation. Theenzyme is allosterically inhibited by the final product, that is by cholesterolitself.
The genetic regulation of cholesterol synthesis has been worked out fairlyrecently, and it operates by quite a neat mechanism. Several membrane proteinsparticipate in it (Figure 11.6a): The Sterol Response Element Binding Protein(SREBP), the SREBP Cleavage Activating Protein (SCAP), and two SREBP-specificproteases (S1P and S2P). SCAP is the actual cholesterol sensor that changesconformation in response to varying cholesterol concentrations within the ERmembrane. If the level of cholesterol is high, SCAP does not bind SREBP, andSREBP is left behind when SCAP relocates from the ER to the Golgi apparatus(Figure 11.6b). Nothing further happens in this case. The action starts whenthe concentration of cholesterol in the ER membrane is low (Figure 11.6c):
1. SCAP adopts a different conformation that now binds SREBP, dragging italong for the ride to the Golgi apparatus.
2. In the Golgi, S1P and S2P lie in ambush fo SREBP and cleave it.
3. The major SREBP fragment is now free to leave the membrane and totranslocate to the nucleus.
5. . . and, if we are to believe current news, cancer, depression, earthquakes, and about 98% ofall other evil befalling mankind.
150 11 Cholesterol metabolism
Golgi
SCAPSREBPS1P
ER
Golgi
ER
NM
SRE
HMG-CoA reductase mRNA
RNA polymerase
+
a)
b)
c)
Figure 11.6 Transcriptional regulation of cholesterol synthesis. a: The elementsthat participate in it. SRE: Sterol response element; SREBP: SRE binding protein; S1P:SREBP-specific protease 1; SCAP: SREBP cleavage activating protein. b: Situation at highcholesterol concentrations. c: The processes occurring at low cholesterol concentration.See text for details.
11.9 Cholesterol in atherosclerosis 151
OOH
OHCOOH
OH
OHCOOH
Figure 11.7 Structure of lovastatin, an inhibitor of HMG-CoA reductase (left) and ofmevalonate (right).
4. In the nucleus, SREBP binds to sterol response elements (SREs). These areshort specific DNA sequences that occur in various places of the genome.
5. One SRE is located upstream of the gene of HMG-CoA reductase. Bindingof SREBP to this element increases transcription and therefore the overallactivity of HMG-CoA reductase.
Induction of HMG-CoA reductase will increase the rate of cholesteorl synthe-sis, which will ultimately suppress the further proteolytic activation of SREBP.
11.9 Cholesterol in atherosclerosis
The level of plasma cholesterol, and in particular LDL-associated cholesterol, isone of the main risk factors of atherosclerosis, the degenerative vascular diseaseunderlying myocardial infarction and stroke. In western countries, this is theleading cause of death, being more common than all cancers and leukemiascombined. Briefly, atherosclerotic lesions develop as follows:6
1. Small mechanical lesions in the vascular endothelium—the innermostlayer of blood vessel walls—allow leakage of blood plasma into the muscularlayers beneath. Formation of these small leakages is thought to be promotedby high blood pressure.
2. The plasma lipoproteins that leaked into the tissue are degraded. Becauseof its low solubility, cholesterol that is released from degraded lipoproteinsprecipitates.
3. The cholesterol particles trigger invasion of phagocytic cells and in thisway contribute to triggering inflammation, which in turn increases the tissuedamage and turns the small, potentially reparable defects of the vessel wallinto large lesions.
A crucial feature of the atherosclerotic lesion is the erosion of the endothe-lium, which normally inhibits blood coagulation. Stroke or infarction occurswhen blood coagulates at the site of such a lesion. Coagulated blood clogs theartery and suffocates the tissue downstream.
6There are other contributing factors, possibly including infectious agents.
152 11 Cholesterol metabolism
The main factors that may induce a high plasma cholesterol concentrationare (1) the diet: Rich in cholesterol, or in fat or excess carbohydrates, which maybe converted first to acetyl-CoA and then to cholesterol, (2) physical inactivity:Sedentary lifestyles, such as typical of prison or university inmates, (3) geneticfactors, in particular a defect of the LDL receptor.
The latter condition is particularly serious if it is homozygous, that is if thereceptor is completely absent. The levels of LDL and cholesterol in the plasmacan reach several times the normal level. The rate of progress of atherosclerosisand the risk of stroke and myocardial infarction are greatly increased, so muchso that the typical life expectancy of homozygous patients is only 30-40 years.Heterozygous patients still are at significantly higher risk than the averageperson, but the condition is more tractable with the available means of therapy.
11.10 Therapy of hypercholesterolemia
The obvious way of treating the various forms of hypercholesterolemia is to tryand lower the blood cholesterol level. What therapeutic principles are availableto do this? Several:
1. Limiting the dietary intake of cholesterol. Since cholesterol occurs onlyin animals but not plants, an obvious way of restricting intake of cholesterol isor reduction of meat and eggs in favour of fruit and vegetables.
2. Vegetables are also rich in poly-unsaturated fatty acids (those with sev-eral C=C double bonds) and in carbohydrate fibers, which seem to have agreater favourable effect than restriction of cholesterol itself. The mechanismby which these beneficial effects arise are not well understood.
3. Inhibiting the intestinal uptake of cholesterol. This can be done withsitosterol, a plant sterol that has no role in human metabolism. The mecha-nistic aspects of this inhibition are not clear but it appears to be some kind ofcompetition, which in turn suggests a specific uptake mechanism for choles-terol. Uptake can also be inhibited by ezetimibe and other recently developeddrugs. The target protein in the intestinal membrane that ezetimibe binds tohas been identified. It is known as the Niemann-Pick C1-like protein 1 (NPC1L1).Ezetimibe interferes with the endocytosis of this protein; it may be that uptakeof cholesterol depends on this same endocytotic process.
4. Inhibition of endogenous cholesterol synthesis. The most important classof synthesis inhibitors are the statins, for example lovastatin, which mimicmevalonate in structure and block the active site of HMG-CoA reductase (Figure11.7).
5. Promotion of cholesterol elimination, by way of bile acid depletion. Asstated above, bile acids are derived from cholesterol. Out of the consider-able amount of bile acids secreted—several grams per day—approximately95% are taken up again in the terminal ileum (the lowermost section of thesmall intestine) and recycled. Cholestyramine is a granulated polymer that
11.10 Therapy of hypercholesterolemia 153
exposes hydrophobic and cationic binding sites, which combine avidly with thehydrophobic and anionic moieties of bile acid molecules. Ingested cholestyra-mine will bind bile acids and simply take them along down the pipe. To replacethe lost bile acids, the liver will increase their synthesis and therefore depletethe pool of cholesterol.
Apart from bile acids, excess cholesterol itself is also secreted into the bile,where it depends on bile acids to remain in solution. A drawback of cholestyra-mine therapy is that, by depleting bile acids, it promotes the precipitation ofcholesterol, favouring the formation of cholesterol bile stones.
While for maximum effect the different principles are often combined, inhi-bition of synthesis with statin drugs is the most effective single principle andcurrently has a prominent place in therapy.
Chapter 12
Amino acid metabolism
We have seen before that during digestion in the intestines proteins are bro-ken down to their constituent amino acids. Proteins contain twenty standardamino acids, which are incorporated into them during translation, and severalnon-standard ones that are mostly formed by post-translational modification.These are much less abundant than the standard amino acids and will not beconsidered here.
Amino acids, in human metabolism, have three main usages:1. As building blocks for our own protein synthesis. Animals, including
humans, are essentially parasites and have a lazy synthetic metabolism. Ac-cordingly, we possess synthetic pathways for only 10 out of the 20 standardamino acids. The residual 10 amino acids have to be obtained from the dietand are called the essential amino acids.
2. As a source of energy. Depending on the composition of the diet, thisrole may be very significant. Carbohydrates occur nearly exclusively in plant-derived foodstuffs. Therefore, on a meat-only diet, amino acids become themajor source of glucose.
3. As building blocks for other things such as nucleotides and heme.We will here focus energy metabolism, that is on the degradation of amino
acids. Synthesis will be skipped, except for some simple examples that involveno more than transamination. We only note that nine out of twenty amino acidscannot be synthesized in mammalian cells and therefore have to be obtainedwith the diet. These essential amino acids are histidine, isoleucine, leucine,lysine, methionine, phenylalanine, threonine, tryptophane, and valine. Arginineis synthesized but apparently not in sufficient amounts and often listed as atenth essential amino acid.
154
12.1 Overview of amino acid degradation 155
Pyruvate
Acetyl-CoA
α-Ketoglutarate
Succinyl-CoA
Fumarate
Oxaloacetate
Acetoacetate
Glucose
Propionyl-CoAIle, Met, Thr, Val
Arg, Gln, Glu, His, Pro
Leu, Lys, Phe, Trp, Tyr
Ala, Cys, Gly , Ser
Asn, Asp
Phe, Tyr
Figure 12.1 Overview of amino acid degradation. Glucogenic amino acids that giverise to either pyruvate or TCA intermediates that can be turned into glucose. Ketogenicamino acids give rise to acetoacetate and acetyl-CoA, which do not yield glucose.
12.1 Overview of amino acid degradation
All amino acidsdegradation amino acids contain at least one nitrogen atom,which forms their α-amino group.Nitrogen has no use in energy metabolismand has to be eliminated. There are two key processes in metabolic nitrogenelimination: 1. Transamination removes the α-amino group from one aminoacid and transfers it to α-ketoglutarate. This leads to the accumulation ofglutamate. 2. The release of nitrogen from glutamate and its conversion tourea. This is accomplished by the urea cycle in the liver.
Several amino acids—arginine, asparagine, glutamine, histidine, lysine, pro-line, tryptophan—contain additional nitrogen atoms in their side chains. Forthese, adapter pathways exist that ultimately also feed the nitrogen into theurea cycle.
Removal of nitrogen is typically an early step in amino acid degradation andleaves behind the carbon skeleton. The structure of the latter is different foreach amino acid, and accordingly each amino acid has its specific pathway ofdegradation. However, they can be grouped into two broad classes. As Figure12.1 shows, most amino acids can be converted to intermediates of the citricacid cycle or to pyruvate. These are called glucogenic amino acids, since theycan contribute to the synthesis of glucose (gluconeogenesis). Those that yieldacetoacetate are called ketogenic, since acetoacetate is one of the two ketonebodies (see section 10.4).
What happens to glucogenic amino acids when they are available in excessover the demand for glucose? This may well happen if we are on a very protein-rich diet. One option would be to first convert all substrates to oxaloacetate
156 12 Amino acid metabolism
in the citric acid cycle and then short-circuit gluconeogenesis and glycolysis atthe level of phosphoenolpyruvate:
Oxaloacetate + GTP ---------------------------------------→ PEP+ GDP+ CO2
PEP + ADP ---------------------------------------→ pyruvate+ATP
Although this would seem reasonably straightforward, this pathway is ap-parently not quantitatively important. Instead, the substrates are drained fromthe TCA cycle at the level of malate by malic enzyme:
Malate+NADP+ ---------------------------------------→ pyruvate+NADPH+H+ + CO2
An advantage of this pathway is that it yields NADPH and not NADH, as isthe case in the formation of oxaloacetate by malate dehydrogenase. Excesspyruvate is mostly converted to acetyl-CoA by pyruvate dehydrogenase andused for fatty acid synthesis; malic enzyme provides part of the NADPH neededby fatty acid synthase.
12.2 Transamination
In the degradation of most standard amino acids, an early step in degrada-tion consists in transamination, which is the transfer of the α- amino groupfrom the amino acid to an α-keto acid. There are several different aminotrans-ferases, each of which is specific for an individual amino acid or for a groupof chemically similar ones, such as for example the group of branched aminoacids, which includes leucine, isoleucine, and valine. With all these enzymes,the α-keto acid that accepts the amino group is always α-ketoglutarate (Fig-ure 12.2). Transamination is freely reversible; therefore, both glutamate andα-ketoglutarate are substrates of every single transaminase. If amino groupsare to be transferred between two amino acids other than glutamate, this willstill occur by transient formation of glutamate (Figure 12.2b).
The mechanism of transamination is depicted in Figure 12.4 for alanine, yetis the same with all transaminases. The reaction occurs in two stages:
1. Transfer of the amino group from alanine to the enzyme, which releasespyruvate, and
2. Transfer of the amino group from the enzyme to α-ketoglutarate, whichreleases glutamate.
In Figure 12.4, only the first half-reaction is shown. The second half-reaction isthe exact reversal of the first one; this also implies that the entire reaction isreversible. Overall, the mechanism consists in the first substrate arriving andleaving before the second substrate enters and leaves; this is dubbed a ping
12.2 Transamination 157
COOH
C
CH2
CH2
COOH
O
COOH
CH
CH3
NH2
+
COOH
CHNH2
CH2
CH2
COOH
COOH
C
CH3
O
+
COOH
CHNH2
CH2
CH2
COOH
COOH
C
CH2
CH2
COOH
O
COOH
C
CH3
O
COOH
CH
CH3
NH2
COOH
CHNH2
CH2
COOH
COOH
C
CH2
COOH
O
1st transaminase 2nd transaminase
a)
b)
Figure 12.2 Transamination reactions. a: Glutamate pyruvate transaminase (alsocalled alanine amino transferase) transfers the α-amino group from alanine to α-ketoglutarate, which yields glutamate and pyruvate. b: All transaminases have α-ketoglutarate as one of their substrates. Transfer of amino groups between arbitraryamino and α-keto acids (here: alanine and oxaloacetate) occurs by transient transfer toα-ketoglutarate.
pong bi bi reaction (Figure 12.3). 1 While two different substrates must be usedfor the reaction to have a net effect, it is of course possible for amino acid 1and amino acid 2 to be identical – the reaction will work just fine, but simplyachieve no net turnover.
The reaction mechanism revolves around the coenzyme pyridoxal phos-phate (PLP):
1. At the outset of the reaction, PLP is bound as a Schiff base to the ε-aminogroup of a lysine residue in the active site (Figure 12.4a).
2. The bond between PLP and the enzyme is separated, and PLP forms aSchiff base with the amino acid substrate instead (Figure 12.4b, steps 1 and 2).
3. The liberated lysine residue abstracts the α hydrogen as a proton (step3), and the electron left behind travels all the way down to the nitrogen at thebottom of the PLP ring. PLP is often said to act as an electron sink. This hasthe effect of turning the bond between the α carbon and the α nitrogen into aSchiff base.
1The mind boggles when one tries to figure out what kind of greek nomenclature Old Worldbiochemists could have dreamed up for this behaviour. Maybe something like: Amphiballisticantidromic sequential?
158 12 Amino acid metabolism
E-Lys-N=PLP
amino acid 1 α-keto acid 1
E-Lys-NH2
pyridoxamine
α-keto acid 2
E-Lys-N=PLP
amino acid 2
Figure 12.3 The ping pong bi bi kinetic mechanism of transamination. The twosubstrates arrive and leave one after another; the second half-reaction (dotted bluearrow) is the exact reversal of the first one (solid blue arrow), save that a differentα-keto is used.
4. The Schiff base is hydrolyzed to yield the α-keto acid and the aminoderivative of PLP, called pyridoxamine phosphate (steps 4 and 5).
The PLP in its various forms stays within the active site throughout, evenwhile not bound to the enzyme covalently. As stated above, the second halfreaction is the exact reversal of the first, and a good exercise for you would beto draw the individual steps yourself.
12.3 The urea cycle
While transamination solves the problem of removing the α-nitrogen for theamino acids other than glutamate, there also must be a mechanism to regener-ate the α-ketoglutarate that is converted to glutamate in each transaminationreaction. This is accomplished in the glutamate dehydrogenase reaction, whichreleases the nitrogen as ammonia (Figure 12.7c).
Ammonia is quite toxic, and its levels in the systemic circulation are kept inthe micromolar range.2 The glutamate dehydrogenase reaction runs primarilyin the liver. There, the ammonia is scooped up immediately by the urea cycle,which converts it to the much less toxic metabolite urea. Urea is released intothe circulation and then eliminated through the kidneys. The overall reactionof the urea cycle is:
NH3 +HCO−3 +Aspartate ---------------------------------------→ (NH2)2CO+ fumarate
with the simultaneous expenditure of several molecules of ATP in order tomake this happen.
12.3.1 Reactions in the urea cycle
The urea cycle involves the following reactions (Figure 12.5):
2In liver cirrhosis, one of the main problems is the lacking capability of the liver to detoxifyammonia derived from bacterial metabolism in the large intestine. Antibiotics are used in thiscondition to reduce bacterial growth and ammonia formation.
12.3 The urea cycle 159
CHNH
+
CH2
P
CH3
O
CHO
CH2
Enzyme
CHNH
+
CH2
P
CH3
O
CHNH
+
CH
COOHCH3
NCH2
Enzyme
CHNH
+
CH2
P
CH3
O
CH NH2
+H
CH2
Enzyme
CHNH
+
CH2
P
CH3
O
CH NH
+
CH
COOHCH3
NHH
H+
C COOHCH3
O
NH2
CHNH
CH2
P
CH3
O
CH
C COOHCH3
NH
CHNH
CH2
P
CH3
O
CH
OH
H+
C COOHCH3
N
CHNH
CH2
P
CH3
O
CH
OH
H+
CH2
Enzyme
NH3
+
CH2
Enzyme
NH2
C COOHCH3
N
CHNH
+
CH2
P
CH3
O
CH
H
a)
b)
1
2
3
4
5
Figure 12.4 Mechanismof transamination. a: Struc-ture of pyridoxalphosphate(PLP), free and covalentlybound to the enzyme. b:Details of the mechanism,shown for the first halfreaction only. At the endof this reaction, PLP retainsthe former α-nitrogen, andthe substrate is releasedas an α-keto acid (Figure12.3). The second half ofthe reaction is the exactreversal of this sequence.
160 12 Amino acid metabolism
C O
O
OHNH2 C
O
OH
OP
O
O
OHATP ADP
NH2
NH2CH
COOH
CH2
CH2
CH2
N
C
NH2
CH
COOH
CH
COOH
ATP ADP
C O
O
OH
P
O
O
OHNH3
NH2C
O
OO P
O
OH
NH2CH
COOH
CH2
CH2
CH2
NH2
NH
CH
COOH
CH2
COOH
NH2CH
COOH
CH2
CH2
CH2
NH
C
NH
AMP
NH2 C
NH2
OOH2
NH2CH
COOH
CH2
CH2
CH2
NH2
NH2CH
COOH
CH2
CH2
CH2
NH
C O
NH2A P P P
NH2CH
COOH
CH2
CH2
CH2
NH
C O
NH
AMP
P P
NH2CH
COOH
CH2
CH3
1 1 1 2
33
4
3
5
COOH
Figure 12.5 Reactions of the urea cycle. Enzymes: 1, carbamoylphosphate syn-thetase; 2: ornithine transcarbamoylase; 3: argininosuccinate synthetase; 4: argini-nosuccinase; 5: arginase.
1. The fixation of ammonia by carbamoylphosphate synthetase, which alsouses bicarbonate as a substrate, and two molecules of ATP.
2. The transfer of the carbamoyl group from carbamoylphosphate to theδ-amino group of ornithine, a non-standard amino acid homologous to lysine,by ornithine transcarbamoylase. This reaction yields citrulline.
3. The ligation of aspartate and citrulline to form argininosuccinate, cat-alyzed by argininosuccinate synthetase. This reaction again requires ATP, whichis converted to AMP in the process.
4. The cleavage of argininosuccinate into fumarate and arginine by argini-nosuccinase.
5. The release of urea from arginine by arginase, which regenerates or-nithine.
Urea is transported in the bloodstream and eliminated by the kidneys.
12.4 Auxiliary reactions in nitrogen transport and elimination 161
Fumarate
Malate
Oxaloacetate
Arginine
Citrulline
ArgininosuccinateOrnithine
Aspartate Glutamate
2 α-Ketoglutarate
αααα-Keto acid 2
Amino acids 1 and 2
Carbamoyl-P
NH3
HCO3-
Glutamate
αααα-Keto acid 1
Urea
Figure 12.6 Cooperation of citric acid cycle, urea cycle, and transamination reactionsin urea synthesis. Typeset in bold are the names of the only metabolites for whichthere is a net turnover. All other metabolites are part of closed cycles.
12.3.2 Role of aspartate in the urea cycle
As evident in Figure 12.5, only one of the nitrogen atoms in the final moleculeof urea enters the cycle as ammonia, whereas the other one is derived fromaspartate. Where does this nitrogen come from, and what becomes of thefumarate generated from this aspartate?
To answer this question, we just need to pull together our previous knowl-edge about the citric acid cycle and about transamination (Figure 12.6). Fu-marate is turned into oxaloacetate in the citric acid cycle, so we can just borrowthese reactions. Oxaloacetate is then transaminated to aspartate; this gets ridof one molecule of glutamate, which acquired its nitrogen by transaminationof another amino acid destined for degradation. In other words, the aspar-tate serves as an intermediate carrier of nitrogen en route from amino aciddegradation to urea synthesis.
12.4 Auxiliary reactions in nitrogen transport and elimi-nation
The urea cycle runs in the liver, yet amino acid degradation runs in manytissues. For example, skeletal muscle has a prominent role in the degradationof branched-chain amino acids. Therefore, a mechanism is required to carrythe nitrogen produced in the peripheral organs to the liver. Ammonia cannotbe used as a carrier since it is too toxic; amino acids are a better alternative.
The most important amino acids that serve as carriers are glutamine andalanine. Glutamine is produced in the peripheral tissues from glutamate byan enzyme called glutamine oxoglutarate γaminotransferase (GOGAT) or—for
162 12 Amino acid metabolism
CH
CH2
CH2
OHO
NH2
O OH
CH
CH2
CH2
OHO
NH2
O OH
C
CH2
CH2
OHO
O OH
O CH
CH2
CH2
OHO
NH2
O NH2
NADH+H+NAD+
+ +
COOH
CHNH2
CH2
CH2
COOH
COOH
C
CH2
CH2
COOH
O
OH2 NH3
NADH+H+NAD+
CH
CH2
CH2
OHO
NH2
O NH2
CH
CH2
CH2
OHO
NH2
O OH
OH2 NH3
a)
b) c)
CH
CH2
CH2
OHO
NH2
O NH2
CH
CH2
CH2
OHO
NH2
O OH
OH2NH3
ATP ADP
d)
Figure 12.7 Auxiliary reactions in nitrogen transport from peripheral tissues to theliver. a: Glutamate synthase (GOGAT) disproportionates glutamate to α- ketoglutarateand glutamine. This happens in the periphery. b, c: Glutamate dehydrogenase (b) andglutaminase (c) release ammonia from glutamate and glutamine, respectively. Thishappens mostly in the liver. d: Glutamine synthetase scavenges excess ammonia. Thisoccurs both in the liver and in peripheral tissues.
the reversal of this reaction—glutamate synthase. Note that this enzyme pro-duces an amide group, not an amine. The second product of this reaction isα-ketoglutarate, which can participate in another round of transamination. Glu-tamine travels to the liver, where its δ-amido group is released as ammonia byglutaminase. The glutamate produced returns to the peripheral tissue (Figure12.8a).3
Alanine is formed from pyruvate by transamination in the peripheral tissues.It travels through the blood to the liver, where the nitrogen is abstracted bytransamination back to pyruvate. Gluconeogenesis then turns pyruvate intoglucose, which is returned to the periphery. This process is called the glucose
3One might think that the glutamate could be further deaminated by glutamate dehydrogenase,and α-ketoglutarate be returned to the periphery. However, the plasma concentration of α-ketoglutarate is very low, so that this does not seem to be quantitatively important.
12.4 Auxiliary reactions in nitrogen transport and elimination 163
GlutamineH2O
Glutamate Urea
Glutamine
NH3
Amino acid
α-Keto acid Glutamate
α-Ketoglutarate
a)
b)
α-Ketoglutarate
Alanine
Glutamate
Ureaα-Keto acid Glutamate
H2O
NH3Pyruvate
Glucose
α-Keto-glutarate
Amino acid
Glucose
Pyruvate
Alanine
1
2
3
1 44 5
Figure 12.8 Organ relationships in nitrogen transport. a: In peripheral tissues suchas skeletal muscle, glutamate is formed by transamination of amino acids being de-graded (1). Glutamine and α-ketoglutarate are formed from two molecules of glutamateby glutamate synthase (2; cf. Figure 12.7a). α- Ketoglutarate takes part in another roundof transamination, whereas glutamine travels to the liver. There, its amido nitrogen isreleased by glutaminase (3; Figure 12.7c), and it returns as glutamate. b: The glucose-alanine cycle combines amino acid degradation and glycolysis in peripheral tissueswith gluconeogenesis in the liver. Pyruvate formed in the periphery is transaminatedto alanine, which is transported to the liver. 4: Alanine aminotransferase; 5: glutamatedehydrogenase.
alanine cycle (Figure 12.8b).
Nitrogen transport via glutamine does not cost any ATP, since GOGAT doesnot require it. In contrast, the glucose alanine cycle is quite costly – for each
164 12 Amino acid metabolism
glutaminase / glutamate dehydrogenase: NH3 ↑
NH3 is high -urea cycle runs at speed
glutamine synthetase:NH3↓
Portal vein / liver artery
To systemic circulation
Figure 12.9 Distribution of enzyme activities related that release or capture ammoniawithin the liver lobule. Ammonia is released from glutamine and glutamate in theperiphery of the lobule, which increases throughput of the urea cycle. Remainingammonia is scooped up and stored away as glutamine before the blood re-enters thegeneral circulation.
nitrogen transported, two ATP molecules are expended, since each round ofgluconeogenesis and glycolysis transports two nitrogens and consumes 4 mo-lecules of ATP.
12.4.1 Auxiliary enzyme activities in urea synthesis and their distri-bution within the liver lobule
The toxicity of ammonia requires its concentration to be kept very low in thesystemic circulation. On the other hand, for the urea cycle to run at speed,the concentration must be high enough to saturate the initial enzyme, car-bamoylphosphate synthetase, to a useful degree. Therefore, ammonia mustbe released as the blood enters the liver tissue, and scooped up again beforethe blood is drained away into the general circulation. To make this work, theenzymes that release ammonia or fix it are strategically distributed in the livertissue (Figure 12.9). We had seen before that the liver has a honeycomb-likefine structure, with the blood filtering through the individual hexagonal lobulifrom the periphery towards the center (Figure 1.10). Glutaminase and gluta-mate dehydrogenase, which release ammonia, are found predominantly in theperiphery of the lobule. This results in an increased concentration of ammoniain the bulk of the lobule tissue, where the urea cycle can therefore run at speed.Scavenging the remaining ammonia is the job of glutamine synthetase, which ismainly found around the central veins of the lobule. This illustrates nicely how
12.5 Degradative pathways of individual amino acids 165
CH
CH2
OHO
NH2
O NH2
CH
CH2
OHO
NH2
O OH
OH2 NH3
Figure 12.10 The conversion of asparagine to aspartic acid by asparaginase. Notethat this is entirely analogous to the glutaminase reaction (Figure 12.7c).
biochemical and anatomical levels of organization are interrelated, and howthe body is not just a bag of cells, not even at the level of individual organs.
Glutamate dehydrogenase is credited with both release and fixation of am-monia. Interestingly, this enzyme can utilize both NAD and NADP as cosub-strates. The former is present in the cell mostly in its oxidized form, that isas NAD+, favouring release of ammonia, whereas the NADP mostly occurs asNADPH, favouring ammonia fixation. It is not clear what regulatory mecha-nisms prevent this from going back and forth in the same cell, which woulddissipate of the difference in the redox states of NAD and NADP.
12.5 Degradative pathways of individual amino acids
For some amino acids the degradation is a very straightforward issue. Forexample, with alanine, aspartate and glutamate, transamination is all that isrequired to turn them into mainstream metabolites. Glutamine and asparaginecan be accommodated by one preceding deamidation, catalyzed by glutaminase(Figure 12.7b) and asparaginase, respectively.
Asparagine is not an essential amino acid, meaning that it can be synthe-sized by human cells; the enzyme responsible for this, asparagine synthetase,uses glutamine as the amide group donor. However, in some forms of leukemia,the leukemic cells are apparently short of synthetic capacity for asparagine.This can be exploited for therapy: Patients are treated with intravenous ap-plication of asparaginase. 4 This lowers the serum level of asparagine andtherefore starves the leukemic cells. However, this is only one component inthe therapy – cytostatic drugs have to be added to make the therapy effective.For the other amino acids, degradation is more elaborate. We will now considera few examples.
4The enzyme is purified from the bacterium Escherichia coli. In healthy patients, injectionof a bacterial protein would rapidly induce antibodies, which would quickly render the enzymeinactive. However, in leukemia patients, the disease itself and the cytostatic drugs simultaneouslyapplied conspire to suppress antibody formation. If it occurs anyway, enzyme prepared fromanother bacterium, Erwinia chrysanthemi, can be used. Immunogenicity of the enzyme can bereduced by derivatization of the protein with polyethylenglycol (PEG).
166 12 Amino acid metabolism
Figure 12.11 Alternate pathwaysof serine catabolism. a: Serine dey-dratase directly produces pyruvateby eliminating ammonia. b: Serinehydroxymethyltransferase removesthe side chain carbon as formalde-hyde and immediately captures itonto tetrahydrofolic acid (THF). Theremainder is glycine. c: Transamina-tion to hydroxypyruvate, reductionto glycerate, and phosphorylation to3-phosphoglycerate provide an alter-native shunt to glycolysis that doesnot produce free ammonia.
CH
CH2
OHO
NH2
OH NH3 CH3
OHO
O
a)
b)
CH
CH2
OHO
NH2
OH CH2
OHO
O
OH
CH
CH2
OHO
OH
OH
CH
CH2
OHO
OH
O P
aKG Glu
NADH+H+
NAD+
ATPADP
c)
CH2 COOH
NH2
C COOHCH2
NH3
+
O
H
H CH2O +
THF
N,N'-methylene-THF
12.5.1 Degradation of serine and glycine
Both serine and glycine are non-essential amino acids, that is they can be bothsynthesized and degraded in human metabolism. The formation of glycineactually occurs in one of the degradation pathways for serine. These two aminoacids are interesting because their degradation is the prime source of single-carbon groups in metabolism. These ‘C1-units’ are bound to the coenzymetetrahydrofolic acid, which then donates them in a variety of reactions, forexample in the biosynthesis of nucleotides. For serine, there are several routesof utilization (Figure 12.11):
1. The most straightforward one consists in its conversion to pyruvate byserine / threonine dehydratase (Figure 12.11a). Note that in this case the nitro-gen is released as ammonia but not by transamination. The pathway thereforepreferably occurs in the liver, which can dispose of the ammonia. The enzymeuses the same coenzyme (pyridoxalphosphate) and a similar mechanism as thetransaminases (Figure 12.13).
2. Serine hydroxymethyltransferase cleaves serine into glycine and formalde-hyde. Formaldehyde, which is highly reactive, is not released by the enzymebut is immediately fixed onto the cosubstrate tetrahydrofolic acid.
3. An alternative pathway transaminates serine to hydroxypyruvate, whichis then reduced to glycerate and phosphorylated to 3-phosphoglycerate. Whileboth pyruvate and 3-phosphoglycerate can serve as substrates for gluconeoge-
12.5 Degradative pathways of individual amino acids 167
CH2 COOH
NH+
CHNH
+
CH2
P
CH3
O
CH
CH2 COOHNH2
C COOHCH2
NH+
CHNH
+
CH2
P
CH3
O
CH
O
H
H
CH2
Enzyme
NH2
CH COOH
NH+
CHNH
CH2
P
CH3
O
CH
CH2O
CH2
Enzyme
NH2
+H
CH2
Enzyme
NH2
OH2
E-PLP
N
N
N
NH
OH
NH2
N C
O
NH
CH
COOH
CH2
CH2
C
O
OHCH2
n
OH2
N
N
NH
NH
OH
NH2
NH
C
O
NH
CH
COOH
CH2
CH2
C
O
OH
n
N
NH
NH
HO
CH2
N
NH
N
OH
CH2
H
a)
b)
Figure 12.12 Catalytic mechanism of serine hydroxymethyltransferase. The reactionproceeds in two steps. a: PLP-dependent cleavage of serine to glycine and formalde-hyde. b: Capture of formaldehyde by tetrahydrofolic acid (THF). In the resulting N,N’-methylene-THF, the mobilizable methylene group is held by two nitrogen atoms as bya pair of tweezers.
nesis, this pathway avoids the release of free ammonia.
It is interesting to note that all the three routes of serine degradation in-volve enzymes that contain pyridoxalphosphate (PLP) as a coenzyme. This isobvious for the third route, since it starts out with transamination. For serinedehydratase and serine hydroxymethyltransferase, this is illustrated in Figure12.12 and Figure 12.13. Please stare at these figure and at Figure 12.4 until younotice the similarities in the roles of PLP in all three reactions. In all cases, thereversible transfer of an electron to the coenzyme destabilizes the substrate
168 12 Amino acid metabolism
C COOHCH2
NH+
CHNH
+
CH2
P
CH3
O
CH
C COOHCH2
NH2
CH2
Enzyme
NH2
+
H
C COOHCH2
NH+
CHNH
+
CH2
P
CH3
O
CH
OH
H
CH2
Enzyme
NH2
NH3
OH2
E-PLPH+
C COOHCH3
NH
OH2
C COOHCH3
O
C COOHCH2
NH+
CHNH
CH2
P
CH3
O
CH
OH
OH2
CH2
Enzyme
NH2
Figure 12.13 Catalytic mechanism of serine dehydratase. While the overall reactionconsists in the elimination of ammonia, the enzyme itself actually eliminates water,which is reflected in its name. The aminoacrylate released by the enzyme is thenspontaneously hydrolyzed to pyruvate and ammonia.
and primes it for the reaction; the specific bond within the substrate that isbroken is then determined by the point of attack of some specific side chainswithin each enzyme’s active site.
We have already seen that glycine is a product of serine hydroxymethyl-transferase. Since that enzyme reaction is reversible, glycine can actually beconverted to pyruvate or 3-phosphoglycerate via serine and thus serve as asubstrate in gluconeogenesis. The required additional carbon, in the form ofN,N’-methylene-THF, can be provided by the glycine cleavage system, an enzymethat completely breaks down glycine (Figure 12.14). Therefore, two glycinesare required to provide one pyruvate. The glycine cleavage system again usespyridoxalphosphate in its catalytic mechanism, and as far as I can see the PLPagain functions in a similar way as described before.
12.5.2 Degradation of leucine
Leucine, isoleucine and valine are collectively referred to as the branched-chainamino acids. One special thing about these is that their degradation proceedslargely in skeletal muscle. This is similar to the degradation of fatty acids,which also occurs to a large extent in muscle, and indeed several steps inleucine degradation have similarity with the reactions we have seen in fattyacid metabolism. Leucine degradation involves the following reactions (Figure12.15):
12.5 Degradative pathways of individual amino acids 169
glycine
glycineNH3+CO2
NAD+
NADH + H+
THF
methylene-THF
serine pyruvate
NH3
Figure 12.14 Glycine catabolism. The glycine cleavage system completely breaksdown glycine, extracting one methylene group as N,N’-methylene-tetrahydrofolate. Thismethylene group can be used by serine hydroxymethyltransferase to convert a secondglycine molecule to serine and then to pyruvate.
1. Transamination by branched chain amino acid (BCAA) transaminase yieldsα-ketoisocaproate.
2. α-Ketoisocaproate undergoes decarboxylation and dehydrogenation byBCAA dehydrogenase. Like the transaminase in step 1, this dehydrogenase isactive with all branched chain amino acids (valine, leucine, isoleucine). The re-action and the organization of the enzyme is completely analogous to pyruvatedehydrogenase and α-ketoglutarate dehydrogenase, and all use the very sameE3 subunit.
3. The resulting metabolite, isovaleryl-CoA, is similar in structure to a fattyacyl-CoA. It likewise undergoes a FAD-dependent dehydrogenation reaction (byisovaleryl-CoA dehydrogenase), which yields isopentenyl-CoA.
4. Biotin-dependent carboxylation yields methylglutaconyl-CoA. In all of theprevious carboxylation reactions we saw, a vicinal carbonyl group facilitatescarboxylation by withdrawing electrons from the carbon that gets carboxylated.This electron-withdrawing effect can be relayed by a conjugated C−−C doublebond, which is the case here.5
5. Addition of water by methylglutaconyl-CoA hydratase yields HMG-CoA.6. HMG-CoA is split by HMG-CoA lyase to acetyl-CoA and acetoacetate, as
seen before in ketone body synthesis. Evidently, leucine is a purely ketogenicamino acid.
12.5.3 Degradation of phenylalanine
While the degradation of leucine shows a comforting similarity to previouslyencountered pathways, the situation is profoundly different with phenylalanine.This is due to the aromatic nature of the side chain. Aromatic rings are quitestable, and therefore some brute force is needed to crack them open. The besttool for this task is molecular oxygen, and lots of it is used in the breakdownof phenylalanine.
5The mediation of electron-withdrawing effects by conjugated C=C double bonds is referredto as the vinylogous effect.
170 12 Amino acid metabolism
CH
CH2
COOH
NH2
CHCH3 CH3
C
CH2
COOH
O
CHCH3 CH3
C
CH2
S
O
CoA
CHCH3 CH3
C
CH
S
O
CoA
CH3 CH3
C
CH
S
O
CoA
CH3 CH2
COOH
C
CH2
S
O
CoA
C
CH3
CH2
COOHOH
C
CH3
S
O
CoA
C
O
CH2
COOHCH3
α-KGGlu
CoA CO2
NAD+
NADH+H+
FADFADH2
CO2
ATP
ADP+Pi
H2O
1 2 3
4
56
Figure 12.15 Degradation of leucine. The reactions are numbered in accordance withthe descriptions in the text.
The degradation of phenylalanine involves the following reactions (Figure12.16):
1. The hydroxylation of phenylalanine to tyrosine by phenylalanine hydrox-ylase. In this reaction, the second oxygen atom is released as water, reduced atthe expense of the redox cosubstrate tetrahydrobiopterin.
2. Transamination of tyrosine yields p-hydroxyphenylpyruvate.3. Advanced magic that uses another oxygen molecule (and ascorbic acid,
vitamin C, as a cofactor) turns hydroxyphenylpyruvate into homogentisate. Ihave traced the bits and pieces in this reaction by highlighting them in coloror boldface, but an actual explanation of the mechanism is beyond my feeblepowers of understanding and narration.
4. Ring cleavage involves a third molecule of oxygen. The product is maley-lacetoacetate.
5. Maleylacetoacetate is cis-trans isomerized to fumarylacetoacetate.6. Fumarylacetoacetate is hydrolyzed to fumarate and acetoacetate.Fumarate can be utilized in gluconeogenesis, whereas acetoacetate cannot.
Whether this means that phenylalanine is both glucogenic and ketogenic, orjust glucogenic because it can contribute to gluconeogenesis, is a purely verbalquestion that we will leave unanswered.
12.6 Hereditary enzyme defects in amino acid metabolism
Since there are as many pathways as there are individual amino acids, it isunderstandable that many of the known inborn errors of metabolism are relatedto amino acid metabolism. We will consider but a few examples that affect thepathways discussed here.
12.6 Hereditary enzyme defects in amino acid metabolism 171
NH2 CH
CH2
COOH
CH
CHCH
CH
CH
NH2 CH
CH2
COOH
CH
CH CH
CH
OH
Biopterin-H4
Biopterin-H2
O2H2O
OC
CH2
COOH
CH
CH CH
CH
OH
CH
CH
CCH2
CCH2
C
O O O
OH
O
OH
CH2
CCH2
O
HOH
O
CCH
CH
C
O
OHOH
O
OH
CH
CH CH
OH
CH2
COOH
AscorbateAscorbate-H2
O2H2OCO2
O2
OH
CH
CH
CCH2
O
CH2
COOHO
O
α-KG
Glu
H2O
1
2
3
4
5
6
Figure 12.16 Degradationof phenylalanine. Enzymes:1, Phenylalanine hydroxylase;2, tyrosine transaminase;3, p-phenylpyruvate dioxy-genase; 4, homogentisatedioxygenase; 5, maleylace-toacetate isomerase; 6,fumarylacetoacetase.
12.6.1 Phenylketonuria
Phenylketonuria is the most common hereditary enzyme defect, at least amongEuropeans. It is clinically manifest in about one among ten thousand persons.Considering that only homozygous people are clinically affected, this works
out to a heterozygote frequency of√
410000 =
150 , that means one in fifty persons
carries a copy of the defective gene and can potentially have children with thisdisease.
The enzyme affected is phenylalanine hydroxylase, the first enzyme in thedegradative pathway (Figure 12.16). The name of the disease stems from thefact that phenylpyruvate and some derivatives thereof are found in the urine.Formation of phenylpyruvate is due to the buildup of phenylalanine, which will
172 12 Amino acid metabolism
Phe Tyr Hydroxyphenyl-pyruvate
Tyrosine-aminotransferase
Phenylalanine hydroxylase
Phenylpyruvate OC
CH2
COOH
CH
CHCH
CH
CH
neurotoxicmetabolites
Figure 12.17 Metabolic derailments in phenylketonuria. See text for details.
eventually cause it to overcome the high KM of tyrosine transaminase (Figure12.17). Phenylpyruvate is believed to give rise to neurotoxic metabolites, al-though the exact nature of these metabolites is not clear. Symptoms includeretardation of neurological and mental development.
The treatment of phenylketonuria is pretty straightforward: Limitation ofdietary phenylalanine. Tyrosine is plentifully available in a reasonably protein-rich diet, so that the lack of endogenous formation won’t be a problem. Thechallenge then is to diagnose the disease in newborn kids, before any damageis done. Happily, the enzyme defect does not cause a problem during fetaldevelopment, since both useful and potentially harmful metabolites are con-stantly equilibrated between the maternal and the fetal circulation. Buildupof a metabolite in the fetus will therefore not occur as long as the mother’smetabolism is able to degrade it.
The modern test for phenylketonuria is boring: Just draw a sample of bloodand determine the phenylalanine concentration in the serum by HPLC. Theoriginal test—the Guthrie test—was a bit more roundabout in principle, yetingenious, exceedingly simple and cheap in practice. Moreover, it nicely illus-trates the power of bacterial genetics in biochemistry, and it therefore meritsinclusion here.
In contrast to mammalian cells, the bacterium Escherichia coli can synthe-size all 20 standard amino acids, as long as it has ammonia, some inorganicsalts, and an organic carbon source such as glucose. Such a mixture constitutesa minimal medium. The Guthrie test makes use of a mutant E. coli strain thatis Phe – , which means that it cannot synthesize phenylalanine on its own. Thisstrain can be grown on a medium that supplies phenylalanine; however, onminimal medium, it will not grow. Here is how it works (Figure 12.18):
1. A petri dish with minimal medium is streaked with the Phe- strain. Thecells will be thinly spread everywhere, but they will not grow for lack of pheny-lalanine.
12.6 Hereditary enzyme defects in amino acid metabolism 173
... we place a our test strips soaked with blood onto the agar. Phe diffuses into agar…
… and creates a zone of bacterial growth around the sample from an afflicted child
Minimal medium (no phenylalanine):No growth of Phe- strain
Rich medium (contains phenylalanine):Growth of Phe- strain
Spread Phe- strain evenly on thesurface of minimal medium:No growth will occur unless..
Figure 12.18 Principle of the Guthrie test for phenylketonuria. See text for additionaldetails.
2. A small piece of filter paper, soaked with a drop of test blood, is placedon the surface of the agar.
3. Small molecules including amino acids will diffuse from the filter paperinto the surrounding agar. If the test sample contains a high level of pheny-lalanine, as is the case with infants that suffer from phenylketonuria, bacterialcells right next to the filter paper will gobble it up and start growing.
4. If very little phenylalanine is available, as will be the case with healthychildren, no bacterial growth will occur around the samples.
Therefore, a zone of bacterial growth around the filter paper snippet willidentify a sample from a patient with phenylketonuria. Note that, for this testto work, we cannot collect the blood sample right away after birth. In thewomb, the fetal blood equilibrates with the mother’s, and so the phenylalanine
174 12 Amino acid metabolism
concentration is only slightly increased (which is good, since it protects thefetus from the damage). We therefore have to allow about 1-2 weeks afterdelivery for phenylalanine to accumulate in the child’s blood. This is one ofthe drawbacks of the Guthrie test in comparison to the HPLC method. Thelatter is more quantitatively accurate and readily detects the smaller increasein phenylalanine concentration observed at the time of delivery.
12.6.2 Tyrosinemia type I
This tyrosinemia is another metabolic defect in the degradation of phenylala-nine and tyrosine. It is very common in parts of Quebec, which is probably re-lated to the original immigration of a comparatively small group of settlers. Thedeficient enzyme is fumarylacetoacetase. All preceding metabolites are backedup (Figure 12.19a). Severe liver damage is prominent among the symptoms;this is ascribed to the toxic effect of maleylacetoacetate, which reacts with DNAand thereby causes damage to it. Therapy is possible with the enzyme inhibitornitro-trifluoromethyl-benzoyl-cyclohexanone (Figure 12.19b). This compoundis structurally similar to both ascorbic acid and p-hydroxyphenylpyruvate, thecosubstrate and the substrate of p-hydroxyphenylpyruvate dioxygenase. Inhi-bition of this enzyme relieves backup of all toxic downstream metabolites. Toreduce the elevated concentration of tyrosine, which can still lead to neurologi-cal deficits, dietary restriction of that amino acid and of phenylalanine is alsorequired.
12.6 Hereditary enzyme defects in amino acid metabolism 175
Tyrosine ↑↑↑↑
p-Hydroxyphenylpyruvate ↑↑↑↑
Homogentisate
Maleylacetoacetate
Fumarylacetoacetate
Fumarate + acetoacetate
p-Hydroxyphenylpyruvate dioxygenase
O CF3
NO2OO
(Nitro-trifluoromethyl-benzoyl)-cyclohexanone (NTBC)
Tyrosine ↑↑↑↑
p-Hydroxyphenylpyruvate ↑↑↑↑
Homogentisate ↑↑↑↑
Maleylacetoacetate ↑↑↑↑
Fumarylacetoacetate ↑↑↑↑
Fumarate + acetoacetate
fumarylacetoacetase
Toxic effect (liver damage)
a)
b)
Figure 12.19 Tyrosinemia type I. a: Enzyme defect and resulting metabolic backupin in tyrosinemia type; b: Therapy with the enzyme inhibitor nitro-trifluoromethyl-benzoyl-cyclohexanone (NTBC).
Chapter 13
Hormonal regulation of metabolism
Metabolism is regulated at different organizational levels. With regulatorymechanisms that operate entirely within the individual cell, the main purpose ishousekeeping, that is to adjust the throughput of catabolic pathways such as tokeep up a ready supply of ATP, NADPH and so on. This is accomplished largelyby feedback inhibition and feedforward activation by key metabolites such asATP and acetyl-CoA. In contrast, hormonal control is about the obligations ofa cell to the organism as a whole. Hormones are released in response to theprevailing metabolic situation of the body. Key examples are the secretion ofinsulin in response to high blood glucose levels, and the secretion of glucagon inresponse to low glucose levels. Hormones may also be secreted in anticipationof an imminent change of the metabolic situation, as is the case with the ’fight-and-flight’ hormones epinephrine and norepinephrine. Other hormones withmajor effects on energy metabolism are the glucocorticoids (cortisone andhydrocortisone) and the thyroid hormones (tri- and tetraiodothyronine).
13.1 Insulin
The insulin most widely known hormone in metabolic regulation is certainlyinsulin, which facilitates the utilization of glucose. This hormone is so namedbecause it is secreted in the pancreatic islets (insula = latin for island, islet).These islets are small portions of tissue that are found interspersed within the
176
13.1 Insulin 177
Trypsin, Insulin
Garbage
b)
a)
c)
d)
Figure 13.1 Pancreatic islets, and thepurification of insulin from them. a: Anislet (marked by arrows) within the ‘redsea’ of exocrine pancreas. Reproduced,with permission from Roger Wagner,from http://www.udel.edu/Biology/Wags/histopage/histopage.htm.b: Initial attempts to purify insulin frompancreas homogenates failed, becausethe hormone was rapidly degraded bythe proteases released from the exocrinecells. c: Banting and Best managedto purify insulin by first occludingthe pancreatic ducts of experimentalanimals. This led to the degeneration ofthe exocrine pancreas, while the isletswere left intact. d: Frederick Banting(left) and Charles Best.
sea of the exocrine pancreas. The exocrine1 pancreas, which accounts for 98%of the entire mass of the organ, is responsible for secreting all the enzymes andbicarbonate we have encountered before. The islets, in contrast, are endocrineand secrete a variety of peptide hormones into the blood stream, most notablyinsulin and glucagon.
The importance of the pancreas in the regulation of blood glucose was rec-
1Exocrine means that the secretion is targeted to the ’outside’ world (and that would includethe intestinal lumen), whereas endocrine secretion is targeted into the blood stream. Thus, allhormones are secreted by endocrine glands, and the branch of medicine that specializes inhormones, their glands and related diseases is endocrinology.
178 13 Hormonal regulation of metabolism
Figure 13.2 Primary struc-ture of insulin. Top: Aminoacid sequences from cow, pig,and humans. Deviations ofthe animal sequences fromthe human one are high-lighted. The lines indicatedisulfide bonds; one disul-fide bond occurs within the(shorter) A chain, whereas Aand B chain are held togetherby two disulfide bonds. Bot-tom: Processing of insulinprecursors. The two finalproducts (insulin and C pep-tide) are both stored in secre-tory vesicles and secreted to-gether.
InsulinC-peptidePreproinsulin
Signal peptide
Proinsulin
N-GIVQQCCTSICSLLYQLENYCN-C
N-FVNQHLCGSHLVEALYLVCGERGFFYTPKT-C
N-GIVQQCCTSICSLLYQLENYCN-C
N-FVNQHLCGSHLVEALYLVCGERGFFYTPKA-C
N-GIVQQCCASVCSLLYQLENYCN-C
N-FVNQHLCGSHLVEALYLVCGERGFFYTPKA-C
ognized by animal experiments, which showed that removal of the pancreasresulted in diabetes. Attempts to purify the active principle—that is, insulin—failed, however, since after grinding up entire pancreas organs all proteins wererapidly degraded by the proteases released from the exocrine pancreas cells(Figure 13.1a). The bright idea that solved this problem occured to Dr. Fred-erick Banting of the University of Toronto (Figure 13.1d). He took advantageof the previous observation that obstruction of the pancreatic duct inducesself-digestion and destruction of the exocrine pancreas, however somewhatsurprisingly without destruction of the islets. By first occluding the pancreaticduct in the animals and then patiently awaiting the degeneration of the exocrinepancreas, Banting and his student Charles Best were able to purify insulin inintact form and demonstrate its ability to revert diabetic symptoms. In remark-able contrast to modern practice, they very generously refrained from claiminga patent. Instead, they invited everyone to join in and supply the world withinsulin, so that within a short period of time it became available and affordableto diabetics worldwide. One simply must love these guys. Great Canadians, andof course well-deserved Nobel prize winners.2
Insulin became the subject of a second Noble prize when Frederick Sanger at
2Charles Best did not officially receive the Nobel Prize; instead, Banting shared it with JohnMacLeod, another researcher at U of T. However, Frederick Banting declared that Best shouldhave been included and split his share of the prize with him to affirm this point. Thanks toAlastair Brownie for correcting this historical detail.
13.1 Insulin 179
Cambridge managed to develop the first workable methodology for protein se-quencing and to elucidate the primary structure of insulin, which thus becamethe first protein with completely known amino acid sequence. The primarystructure of insulin is depicted in Figure 13.2. It consists of an A chain and aB chain, which are derived from a common precursor that undergoes a seriesof site-specific cleavages. The final products, insulin and the C peptide, areboth stored in secretory vesicles and are released together. Note the sequencevariations between the bovine, the porcine and the human insulin. Becauseof these sequence variations, it is possible for diabetics to develop antibodiesagainst the animal insulins; this is a major reason why nowadays recombinantlyproduced human insulin is preferred for the treatment of diabetes.
13.1.1 Mechanism of secretion of insulin from pancreatic β-cells
Insulin is secreted by a certain cell type within the pancreatic islets, the β-cells(α-cells secrete glucagon, the antagonist hormone of insulin). The secretion iscontrolled by the level of glucose in the blood. The textbook model for thisregulation works as follows:
1. Glucose enters the β-cell and gets degraded, which yields ATP. Thus, thelevel of intracellular ATP varies in proportion to the blood glucose.
2. ATP binds to the sulfonylurea receptor, which is associated with theKir K+ channel. Binding of ATP closes the channel, which depolarizes themembrane.
3. Depolarization opens voltage-gated Ca++ channels and allows Ca++ intothe cell. The increased intracellular Ca++ then triggers insulin secretion byexocytosis.This step is analogous to neurotransmitter exocytosis in nerve cellsin response to presynaptic action potentials.
The sulfonylurea receptor is so named because it is the target of sulfony-lurea derivatives such as tolbutamide (Figure 13.3). These drugs mimic theeffect of ATP on the receptor, thus stimulating the release of insulin from theβ-cells. This is an important therapeutic principle in diabetes type II. We willlearn more about this later.
13.1.2 The insulin receptor and its intracellular second messengersand effectors
The biochemical effects of insulin on its target cells can be summarized in onesimple phrase: Obscenely complex. The intention of the following is to just giveyou a feel of that complexity, but there is no obligation to memorize each andevery detail of it. The insulin receptor is a transmembrane protein located in thecytoplasmic membrane. It has an extracellular domain that specifically bindsinsulin; binding causes activation of the intracellular domain, which is a proteintyrosine kinase (Figure 13.4a). The first protein that gets phosphorylated bythis enzyme is another insulin receptor molecule (Figure 13.4b). This mutual
180 13 Hormonal regulation of metabolism
Figure 13.3 The control ofinsulin secretion. a: Overview.b: Structural sketch of thesulfonylurea receptor (green)and the associated Kir K+
channel. The cylinders repre-sent transmembrane helices.The ATP binding folds (ABF)are on the cytosolic side ofthe membrane. c: Bindingof ATP will cause a confor-mational change to the sul-fonylurea receptor, which inturn will cause the channel toclose. This will partially de-polarize the membrane andinduce insulin secretion viaopening of a voltage-gatedcalcium channel. d: Structureof tolbutamide, an oral drugthat binds to the sulfonylureareceptor and is used in typeII diabetes. The sulfonylureamoiety is highlighted.
b) Kir-Channel Sulfonylurea receptor
ABF ABF
a) Glucose
Glucose
H2O + CO2
ADP+Pi
ATP
K+
Membrane potential
+
Ca++
Insulin
Insulin
ATP ATP
ATP
K+
c)
CH3 S
O
O
NH
O
NH
CH2
CH2
CH2
CH3
d)
phosphorylation of insulin receptors locks them in the active state, from whichthey will only revert to the inactive state upon dephosphorylation by a proteintyrosine phosphatase, even if insulin leaves the receptor again.3
Apart from this mutual phosphorylation, the insulin receptor kinase acts
3However, it seems that insulin stays bound to the receptor for very long periods of time.
13.1 Insulin 181
Receptor domain (extracellular)
Enzyme domain (intracellular)
Ligand
Inactive enzyme Active enzyme
ATP ADPP
P PP
P
ATP ADP
IRS-2(3,4) IRS-2(3,4) P
P
ATP ADP
IRS-1 IRS-1 P
P
P
b)
a)
Figure 13.4 The insulin receptor. a: The receptor is a transmembrane protein thathas an extracellular ligand-binding domain and an intracellular protein kinase domain;the kinase is activated by binding of insulin. b: Ligand-activated insulin receptors willfirst perform mutual phosphorylation and then phosphorylate a series of intracellularregulatory proteins, the so-called insulin receptor substrates (IRS).
upon number of intracellular proteins, which are collectively referred to as in-sulin receptor substrates (IRS; see again Figure 13.4b). In their phosphorylatedstates, these proteins activate a variety of other so-called adapter proteins,which then control various effector cascades. Some important regulatory cas-cades are shown in Figure 13.5:
1. The stimulation of glycogen synthesis. Figure 13.5a shows the cascade
182 13 Hormonal regulation of metabolism
that leads to the activation of glycogen synthase:(a) The receptor phosphorylates insulin substrate 1 (IRS-1).(b) Phosphorylated IRS-1 binds to and activates PIP2-3-kinase, which phospho-
rylates the membrane lipid phosphatidylinositol-bisphosphate (PIP2) to thecorresponding trisphosphate (PIP3).
(c) PIP3 binds to and activates protein kinase B, which in turn phosphorylatesglycogen synthase kinase 3.
(d) In its phosphorylated state, glycogen synthase kinase 3 is less active, whichresults in a lower degree of phosphorylation and therefore higher activityof glycogen synthase.
Note that this is only one of several regulatory mechanisms by which insulinaffects glycogen synthesis. Another one consists in the activation of phospho-diesterase, which will lower the level of cAMP, thereby countering the effects ofepinephrine and of glucagon (see later).
2. The induction of enzymes for glycolysis. This cascade is shown in Figure13.5b:
(a) The first intermediate is again IRS-1. However, in this case IRS-1 it bindsto a different adapter protein, which is called Grb-2.
(b) Grb-2 forms a complex with two other proteins, Sos and Ras; this results inthe exchange of GDP bound to Ras for GTP, and subsequently the activationof a protein kinase called Raf-1.
(c) Two more protein kinases—MEK and MAPK—are phosphorylated in turn.The latter one acts on nuclear transcription factors, which then increasethe rate of transcription of the genes encoding enzymes for glycolysis andfor triacylglycerol synthesis.
3. The increase of cellular glucose uptake. This is accomplished by thetranslocation of GLUT4 transporters from intracellular vesicles to the cytoplas-mic membrane. This translocation is reversible (Figure 13.5c). The cascadecontrolling this translocation again involves IRS-1, PIP3, and protein kinase B(Figure 13.5d).
One question that often comes to mind in this context is: Why did natureinvent all these complicated, lengthy cascades? The standard textbook answeris signal amplification: If each molecule of protein kinase X activates multiplemolecules of kinase Y, each of which in turn activates multiple molecules ofeffector enzyme Z, then the number of final effector molecules can be farlarger than the number of hormone molecules that did bind to the cell. Avalid example for this rationale is glycogen phosphorylase. However, signalamplification is not always a compelling reason. For example, the numberof nuclear transcription factor molecules required to trigger transcriptionalchanges is probably not any larger than the number of insulin receptors onthe cell surface, so no signal amplification is needed in this case. What otherreasons could there be?
13
.1In
sulin
18
3
PKB
I I
P P
PIP2 PIP3
I I
PI-3K IRS-1IRS-1PGlycogen synthase kinase 3 (active)
P-Glycogen synthase kinase 3 (inactive)
Glycogen synthase (active)
P-Glycogen synthase (inactive)
PKA
I I
P P
Grb-2IRS-1 IRS-1 P
Sos
Ras
Raf-1
GTP
GDP
MEK
MEK-P
MAPK-1
MAPK-1-P
Phosphorylation of nuclear factors, induction of genes for glycolytic enzymes etc.
displaces
I Ib)
a)Glucose
GlucoseHigh insulin Low insulin
cytoplasmic membrane
Glucose transporter 4 stored in vesicles
Glucose transporter 4 exposed on cell surface
Phosphorylation of cytoskeletal proteins
Transport of GluT4 to cell surface by vesicle fusion
PKB
I I
P P
PIP2 PIP3
I I
PI-3K IRS-1IRS-1P
d)
c)
Figure 13.5 Intracellular effects of insulin receptor stimulation. a: One of the pathways that lead to activation of glycogensynthesis. b: Mediation of transcriptional effects of insulin. c: Translocation of glucose transporters from intracellular vesiclesto the cytoplasmic membrane. d: Movement of glucose transporters is mediated by protein phosphorylation, too. (See text forfurther details.)
184 13 Hormonal regulation of metabolism
extracellular space
cytosol
αβγ heterotrimeric G-protein
‘7TM’ - receptor
GDP
N
βγ α
GDP
GTP
Ligand
active
αGTP
βγ
N
inactive
αβγ
N
GDP
Pi
Figure 13.6 Function of a G protein-coupled receptor. Upon binding of the ligand tothe extracellular face, a conformational change occurs to both the receptor and the Gprotein bound to it. This leads to the exchange of GDP for GTP on the α-subunit ofthe G protein, which then dissociates from the βγ-dimer and finds its effector protein.When the GTP’ase activity that is built into the α-subunit cleaves GTP to GDP, the α-subunit reverts to its inactive conformation, leaves its target protein, and re-associateswith the β- and γ-subunits and the receptor.
I personally prefer the idea that this complexity serves the purpose of allow-ing different signaling pathways to interact and still yield one coherent, sensibleresponse. The network of intracellular kinases, phosphatases and adapter pro-teins constitutes the brain of the cell, and a brain just needs a certain level ofcomplexity.
13.2 Glucagon and epinephrine
The metabolic effects of glucagon epinephrine are similar to each other butopposite to that of insulin. Glucagon is mainly concerned with metabolic reg-ulation, whereas epinephrine also has pronounced effects on heart rate, bloodpressure and so on. While the two hormones have separate, specific receptors,both of these fall into the group of the G protein-coupled receptors (GPCRs).The workings of a GPCR are illustrated in Figure 13.6. The receptor is a mem-brane protein with seven transmembrane helices, which on its cytosolic faceis associated with a GTP-binding protein, or G protein. This protein has threesubunits; the α-subunit is bound to GDP in its resting state. Signal transductionby a GPCR works as follows:
1. Binding of the ligand to the extracellular face of the receptor causes a con-formational change that involves both the receptor and the heterotrimericG protein.
2. On the G protein, the conformational change leads to the exchange ofGDP for GTP on the α-subunit.
13.2 Glucagon and epinephrine 185
Adenylate cyclase
βγATP
RcAMPR RcAMP
R
C C
cAMP
cAMPcAMP
C CProtein kinase A, inactiveProtein kinase A,active
αGTP
Figure 13.7 Activation of adenylate cyclase and protein kinase A by epinephrine orglucagon. The α-subunit of the G protein binds to adenylate cyclase, which causes itto synthesize cAMP. Protein kinase A, in its resting state, exists as a complex of twocatalytic (C) and two regulatory (R) subunits. Binding of cAMP to the R subunits releasesand thereby activates the catalytic subunits.
3. Upon binding of GTP, the α-subunit dissociates from the βγ-dimer andbinds to its effector protein.
4. After a certain amount of time, the GTPase activity that is built into theα-subunit cleaves GTP to GDP.
5. Cleavage of GTP causes the α-subunit to revert to its inactive conforma-tion, to leave its target protein, and to re-associate with the βγ-dimers.The system thus returns to its resting state.
Both the epinephrine receptor More specifically, the β-adrenergic receptor,of which there again are several subtypes. and the glucagon receptor are cou-pled to the same G protein, which binds to adenylate cyclase as its effectorprotein and activates itadenylate cyclase. Because of this, the metabolic effectson a cell that has receptors for both of them will always be similar. Differencesbetween the effects of the two hormones arise from the fact that the receptorshave different tissue distributions; many cells have receptors for epinephrinebut not glucagon.
The consequences of the stimulation of adenylate cyclase are depicted inFigure 13.7. Protein kinase A, as we have seen before, has several effects:
1. It phosphorylates and thereby inactives glycogen synthase.2. It also phosphorylates phosphorylase kinase, which in turn phosphory-
lates and thereby activates glycogen phosphorylase, so that more glucose getsreleased from glycogen.
186 13 Hormonal regulation of metabolism
Figure 13.8 Nuclearhormone receptors andtranscriptional regulation.a: Structures of cortisol (left)and of the thyroid hormonetriiodothyronine (right). Theiodines contained in thishormone (and the similartetraiodothyronine) are thereason why you need iodinein your diet. b: Simplifiedschematic of the commonmode of function of the twohormones. H: hormone; R:receptor. Of course, there aredifferent, specific receptorsfor the two hormones.
O
CH3
CH3
C O
CH2OH
H H
OH OH
I I
CH2
CHNH2
COOH
O
I
OH
RH
H
RH
mRNA
Proteins(Enzymes, Receptors, …)
b)
a)
3. It phosporylates phosphofructokinase 2, thereby switching on the fructose-2,6-bisphosphatase activity on that bifunctional enzyme. This reduces theconcentration of fructose-2,6-bisphosphate and slows down glycolysis and ac-celerates gluconeogenesis.
4. It phosphorylates and thereby activates hormone-sensitive lipase in fattissue. This leads to the release of glycerol and fatty acids into the blood.
As stated before, all these effects are antagonistic to those of insulin andwill tend to sustain or increase blood glucose levels.
13.3 Glucocorticoids and thyroid hormones
Glucagon and epinephrine act primarily on the activity of pre-formed enzymes(protein kinase A affects transcriptional regulation as well, though). Insulinboth affects pre-formed enzymes and induces the synthesis of new ones. Glu-cocorticoids thyroid hormones mostly act by way of genetic regulation. Theirreceptors are not located at the cell surface but inside the cell. They are eitherfound in the nucleus to begin with, or they translocate from the cytosol tothe nucleus upon binding their ligand. The mechanism of this regulation isdepicted in Figure 13.8 in simplified form. Binding of the hormone (H) to thereceptor (R) causes the latter to recruit a bunch of other proteins (omitted) andtogether with these bind to specific target sequences in the DNA. This changesthe expression levels of genes nearby. The proteins encoded by these genes willinclude enzymes of energy metabolism but also other hormone receptors; for
13.4 Leptin 187
Figure 13.9 Obese mice, which are homozygously deficient for leptin, can be broughtto normal weight by way of leptin substitution. (Image ripped off from http://www.biochemsoctrans.org/bst/033/1063/bst0331063f01.gif.
example, cortisol and cortisone induce enzymes for glycogen metabolism andgluconeogenesis but also receptors for epinephrine. Major effects of cortisolaction are:
1. Stimulation of protein breakdown. The amino acids released are largelyused for gluconeogenesis.
2. Increased enzyme activities for glycogen synthesis and breakdown (!), andfor gluconeogenesis.
3. Increased sensitivity to epinephrine and norepinephrine.4. Suppression of pain and inflammation by inhibition of prostaglandin syn-
thesis.Cortisol is often considered a chronical stress hormone, in contrast to the
acute stress hormones epinephrine and norepinephrine.Thyroid hormones also have a variety of regulatory targets. One key effect is
the induction of thermogenin, which is simply a respiratory chain uncouplingprotein. The mode of action of uncoupling proteins has been described insection 6.1. The up-regulation of uncoupling proteins is responsible for thefinding of accelerated metabolism, hyperthermia and weight loss in patientswith excessive thyroid hormone secretion (hyperthyreosis).
13.4 Leptin
A relatively recently discovered hormone is leptin. This peptide hormone isreleased by fat cells and taken up by the hypothalamus, where it restrictsthe appetite. The hypothalamus has a key role in the homeostasis of manyphysiological parameters and controls the autonomic nervous system as wellas the hypophyseal gland, which in turn controls the activity of the thyroid andadrenal glands. The major variable that controls leptin secretion is simply thevolume of fat tissue, which means that leptin regulates metabolism on a muchlonger time scale than the other hormones discussed above.
The discovery of leptin was accomplished with two different mice strainsthat lack the hormone, named “obese mice”, or the receptor, named “diabetic
188 13 Hormonal regulation of metabolism
mice”. Despite these names, both strains show the same combination of over-weight and type II diabetes. While the receptor-deficient strain unsurprisingly isresistant to leptin, the hormone-deficient one reacts to the hormone in a quiteimpressive fashion (Figure 13.9. Leptin deficiency is not a common conditionin humans, however, so that the therapeutic potential of leptin substitution islimited.
Chapter 14
Diabetes mellitus
To conclude this course, we will try and apply what we have learned to un-derstand the metabolic phenomena underlying the most common metabolicdisease, diabetes mellitus.
The name ‘diabetes mellitus’ means ‘honey-sweet flow-through’.1 That tellsus about two things:
1. An important symptom of the disease: Copious flow of urine, containingsignificant amounts of glucose.
2. A classical method of diagnosis. The alternative to diabetes mellitus isdiabetes insipidus (insipid, flavourless), which is caused by a deficiency of theantidiuretic hormone due to lesions in the posterior lobe of the hypophysealgland and is not related to glucose metabolism.
If you plan on going on to medical school, you will be glad to learn that theclassical method of diagnosis is no longer used in clinical practice.
1For geeks: More precisely, ‘marching through’ – you may recall the term ‘adiabatic’ fromthermodynamics, meaning ‘without permeation’, which is the opposite of ‘diabetic’. The syllablealso occurs in the title of Xenophon’s work Anabasis, or Invasion, which details his experiencesas a Greek mercenary in the ancient Persian empire. This work served Alexander the Great in thepreparation for his campaign into Persia.
189
190 14 Diabetes mellitus
14.1 The role of insulin and the causation of diabetes
The blood glucose concentration is normally very tightly regulated, most no-tably by the two antagonistic hormones insulin and glucagon, such that it iskept between 5 and 9 mM. Insulin is responsible for imposing the upper limiton the blood glucose concentration, and the lack of insulin secretion or of in-sulin effect are the foremost causes of diabetes. There are two major forms ofdiabetes mellitus:
1. Type I is caused by a complete defect of insulin secretion, due to destruc-tion of the β-cells in the pancreatic islets.
2. Type II is caused by a relative insensitivity of the target cells of insulin tothe effects of the hormone.
Therefore, to understand diabetes, it is crucial to understand the physiolog-ical effects of insulin. As noted before, insulin is secreted in times of ampleglucose supply and promotes glucose utilization. Its major metabolic effects are(1) stimulation of glycolysis and inhibition of gluconeogenesis, (2) stimulationof glycogen synthesis and inhibition of glycogen breakdown, and (3) asimula-tion of triacylglycerol synthesis and inhibition of its breakdown.
Insulin also controls the uptake of glucose into the cells of many tissues.There are different types of glucose transporters in cell membranes. The maindistinctions are:
1. Sodium co-transport versus facilitated diffusion (Figure 1.7). Sodiumco-transporters are found where glucose needs to be transported against aconcentration gradient, as is the case in the intestines and the kidney tubules(see below). This transport is always active and never regulated by insulin.
2. Insulin-dependent versus insulin-independent. This disctinction onlyapplies to facilitated diffusion. In insulin-dependent tissues, the hormone con-trols the distribution of glucose transporters between the cytoplasmic mem-brane and an inactive intracellular compartment, as discussed previously (Fig-ure 13.5c and d).
The uptake of glucose is independent of insulin in (1) liver, (2) brain, (3) bloodcells, (4) lens and cornea of the eye. On the other hand, Insulin is required forthe uptake of glucose in muscle, fat and most other tissues. Therefore, sincewithout insulin most cells simply cannot import glucose, it piles up in the bloodof diabetics to very high concentrations. The high blood glucose concentrationis directly responsible for some of the symptoms of diabetes (see below).
14.2 Effects of insulin deficiency on carbohydratemetabolism
Inhibition of glucose consumption in the peripheral tissues is only one effectcontributing to elevated blood glucose levels; equally important is the increaseof glucose formation in the liver.
14.2 Effects of insulin deficiency on carbohydrate metabolism 191
AC
β-AR
Epinephrine
ATP cAMP
IR
Phosphodiesterase 3
AMP
Insulin
Protein kinase A PFK-2 / Fructose-2,6-bisp’ase
Pyruvate dehydrogenase
GlucosePyruvateAcetyl-CoA
PFK-1
Fructose-2,6-bis-P
Fructose-1,6-bisp’ase
GR
Glucagon
AC
β-AR
ATP cAMP
IR
Phosphodiesterase 3
AMP
Insulin
Protein kinase A
Phosphorylase kinase
Glycogen
Phosphorylase
Glucose
Glycogen synthase
Glycogen
P-Glycogen synthase
Glycogen synthase kinase
P-Glycogen synthase kinase
Epinephrine
GR
Glucagon
a)
b)
c)
IR
Insulin
GluT4
Glucose
Glucose
Proteins
ATP
Amino acids α-Keto-acids
α-KG Glutamate
Glutamine
Acetyl-CoA, ATP
To liver (urea cycle / gluconeogenesis)
Pyruvate
Alanine
Figure 14.1 Derailment of glucose and amino acid metabolism in diabetes. a: Glycol-ysis and gluconeogenesis; b: Glycogen metabolism; c: Protein degradation in muscle.See text for details.
192 14 Diabetes mellitus
When insulin is lacking, the insulin-antagonistic hormones glucagon andepinephrine dominate metabolic regulation. This will drive up gluconeogenesisand inhibit glycolysis, which effects are mediated by the intracellular secondmessengers cAMP and fructose-2,6-bisphosphate (Figure 14.1a ; also see Figure7.4). Similarly, glycogen synthesis will be inhibited and glycogen degradationwill be accelerated, by way of the previously discussed intracellular cascadesthat result in the phosphorylation of both glycogen synthase, which is inac-tivated, and phosphorylase, which is activated (Figures 14.1b and 8.5). Therising level of blood glucose is further compounded by the derailment of mus-cle metabolism (Figure 14.1c). Since muscle can no longer take up glucose, itturns to protein breakdown to fill its needs for ATP. Disposal of nitrogen fromdegraded amino acids generates glutamine and alanine, which are transportedto the liver, where they feed the urea cycle and gluconeogenesis as discussedpreviously (Figure 12.8).
14.3 Insulin deficiency and lipid metabolism
In fat tissue, lack of insulin leads to disinhibition of hormone-sensitive lipase(Figure 14.2). This induces breakdown of triacylglyerol to glycerol and fattyacids, which are released into the circulation. Glycerol will be fed into gluco-neogenesis in the liver. Fatty acids may also enter the liver and be degraded toacetyl-CoA. The amount of acetyl-CoA in the liver is further increased by theexcess of blood glucose, which increases the intracellular glucose level (remem-ber that the liver can take up glucose without insulin). This will drive up thesynthesis of ketone bodies, triacylglycerol and cholesterol, the levels of all ofwhich are increased in the blood of diabetic patients. The increased blood fatincreases the incidence and severity of atherosclerosis among diabetic patients.
14.4 Laboratory findings and clinical symptoms in acutetype I diabetes
From the foregoing, we can understand the following laboratory findings inacute type I diabetes:
1. Increased blood glucose. The normal concentration range is 5-9 mM; inacute diabetes, it can be several times higher.
2. Acidic deviation of the blood pH. This is mainly due to the increasedformation of ketone bodies, which are acids. This condition is calledketoacidosis.
3. Increased blood fats (lipoproteins).4. Increased plasma and urine levels of urea, due to the accelerated protein
breakdown in muscle.5. Decreased levels of insulin and of C-peptide.
14.4 Laboratory findings and clinical symptoms in acute type I diabetes 193
AC
β-AR
Epinephrine
ATP cAMP
IR
Phosphodiesterase 3
AMP
Insulin
Protein kinase A
Hormone-sensitive lipase
Triacylglycerol Fatty acids + glycerol
Fatty acids from fat tissue
Glucose
Glucose
Pyruvate
Acetyl-CoA Fatty acids, TAG
Ketone bodies Cholesterol Lipoproteins
a)
b)
Figure 14.2 Derailment of fat metabolism in diabetes. a: Disinhibition of hormone-sensitive lipase in fat tissue triggers breakdown of triacylglycerol. b: Increased levelsof fatty acids and of glucose lead to increased formation of ketone bodies, cholesterol,and triacylglycerol.
Clinically, acute type I diabetes is characterized by these symptoms:
1. Thirst, increased urine flow. Thirst, obviously, is due to the loss of fluid,and this in turn is due to the osmotic activity of glucose in the urine. Thereason why diabetics lose glucose in the urine is discussed below.
2. Acetone smell. As discussed in section 10.4, acetone forms as a byprod-uct of ketone bodies.
3. Recent loss of weight. Both protein and fat are being degraded, and theloss of glucose in the urine can amount to a significant loss of calories.
4. In severe cases, the patients may be unconscious (comatose). Two effectsmay contribute: The pH deviation (ketoacidosis), and blood plasma hyperos-molarity, a consequence of the high glucose concentration. The high osmotic
194 14 Diabetes mellitus
activity of the blood and interstitial fluids drains water from the cells, whichhampers cell function.
5. Some patients report a recent flu-like infection, sometimes with chestpain or even myocarditis (inflammation of the heart muscle).
14.5 The cause of β-cell destruction in type I diabetes
The last symptom in the preceding list is related to the Coxsackie virus infec-tion that results in the destruction of the pancreatic islet β-cells. Coxsackieviruses are small RNA viruses taxonomically related to poliovirus and hepatitisA virus. Only certain Coxsackie virus types are prone to cause diabetes. Themechanism of cell damage consists in an immunological cross-reaction. The im-mune system copes with viral infections by way of destroying the virus-infectedcells. In the case of Coxsackie virus type B4, and possibly some other types,immune cells evoked by the virus mistake surface antigens on β-cells for viralantigens and accordingly destroy them.
The risk of a a person to suffer from diabetes as a consequence of a Cox-sackie type B4 infection is strongly dependent on their genetic background,more specifically their HLA genotype. For example, people with the HLA-DQhaplotype A1: 0301-0302 / B1: 0501-0201 carry a twentyfold increased riskto suffer diabetes, relative to the population average. Another haplotype, B1:0602, has a relative risk of only 3% of the population average. Figure 14.3summarizes the basics of these immunological aspects.
14.6 Why do diabetic patients lose glucose in the urine?
Glucose is precious, and in healthy individuals the concentration of glucose inthe urine is negligible. Why then does glucose appear in the urine in diabeticpatients? To understand this, let’s have a brief look at how the kidney producesurine. The kidney contains a large number of similar functional units, each ofwhich is called a nephron. Within each nephron, urine production proceeds intwo stages (Figure 14.4):
1. Ultrafiltration of the blood plasma. This occurs in the glomerulus; allsmall solutes found in the blood plasma (glucose, ions, amino acids and so on)will appear in the filtrate.
2. Selective, active reuptake of solutes and of water in the tubuli of thenephron.
The capacity of glucose reuptake is limited by the abundance of glucosetransporter molecules in the tubuli. While quite a few processes in metabolismhave a functional reserve, the transport of glucose is not one of them; itscapacity saturates just slightly above the physiological range of the glucoseconcentration in the blood plasma and the primary urine filtrate.
14.6 Why do diabetic patients lose glucose in the urine? 195
T T
T cellreceptor
HLAmolecule
viral peptide
cellularpeptide
high-risk HLAmolecule
a)
b)
c)
Figure 14.3 Immunological causation of diabetes type I. a: Basics of antiviral immu-nity. Each cell presents peptides that result from intracellular protein breakdown onits surface, where they are associated with HLA antigens. If a virus infects the cell, viralpeptides get presented as well. Specific T lymphocytes recognize the viral peptides viatheir T cell receptors, and in response become activated, destroy the cell and propagate.b: The T cell receptor recognizes not just the viral peptide but instead the entire HLA /peptide complex. Some high-risk HLA antigens, when associated with innocent cellularpeptides, may mimic the structure of the viral peptide and thus induce destruction ofa non-infected cell. c, left: Structure of a HLA antigen with a peptide bound. Right: theapproximate outline of the binding site of the T cell receptor is indicated.
196 14 Diabetes mellitus
Blood perfusion (1500 l / day)
Reuptake of glucose and of other solutes by specific transport, of water by osmosis
Collection and excretion (1.5 l/ day)
Filtration (150 l / day)
Amount excreted
Plasma glucose concentration
Reabsorption maximum (10 mM)
Amount filtrated
a)
b)
Figure 14.4 Glucose secretion in the urine in diabetes. a: The nephron and itsfunction. Blood plasma is filtrated in the glomerulus (top), and the filtrate passedthrough the tubuli before excretion. Most of the water and many solutes, includingglucose and amino acids, are reclaimed by active transport in the tubuli. b: Themaximum rate of glucose reuptake is limited by the number of transporter molecules.It is saturated at approximately 10 mM glucose.
14.7 Treatment of type I diabetes
The fundamental thing, of course, is to replace the lacking insulin. An acutely ill,possibly comatose patient will require additional initial measures. Intravenousinfusion therapy is performed, with the following goals:
1. Replacement of fluid,
2. Adjustment of blood pH and electrolytes (potassium, sodium, calcium),which are commonly derailed by the disturbed kidney function,
3. Rapid adjustment of insulin dosage, guided by frequent monitoring of
14.8 Long-term complications of diabetes mellitus 197
blood glucose.
14.8 Long-term complications of diabetes mellitus
When the acute situation has been stabilized, intravenous therapy can be discon-tinued, and insulin can be applied by subcutaneous injection. It is important toadjust the diet of the patient and the insulin therapy so that the blood glucoseremains as close to physiological concentrations as possible. This is necessaryto prevent or postpone the long-term complications of diabetes, which will becaused if blood glucose is chronically elevated:2
1. Increased formation of sorbitol from glucose via the sorbitol pathway(Figure 4.8) in the lens of the eye will induce cataract.
2. Increased conversion of glucose to fat will lead to enhanced blood fatsand promote atherosclerosis.
3. Tissue damage to various organs (peripheral nerves, kidney). The mech-anism is not completely understood but seems to involve direct glucosylationof proteins, and possibly again the sorbitol pathway.
14.8.1 Insulin preparations for substitution therapy
In order to keep blood glucose in a physiological range, it is necessary tomaintain the blood level of insulin be in its physiological range, too. This isnot trivial. Circulating insulin is inactivated by circulating peptidases and has ahalf life of only about 25 minutes. Thus, after intravenous injection of a singledose of insulin, the concentration declines rapidly. In contrast, the pancreassupplies insulin continuously, with peaks after meals and a lower but fairlysteady level during the intervals between them.
In acute therapy and in a hospital setting, the insulin level can be adjustedas needed through continuous intravenous infusion. In outpatients and forsustained therapy, however, we need other methods to mimic or at least ap-proximate the physiological time course.
One way to achieve a more protracted time course of the plasma insulin levelis through subcutaneous application. When insulin is injected subcutaneously,it will wind up in the interstitial space. Insulin then has to “reversely distribute”into the circulation. This uptake is fairly rapid for monomeric insulin, whichis small enough to diffuse through the pores in the capillary wall. However, athigh concentrations, insulin self-associates into hexamers (Figure 14.5), whichare too large for rapid capillary uptake. Only the monomers that remain presentat equilibrium will enter the bloodstream. If the injection is applied shortly
2It is interesting to note that the tissues most severely affected by diabetic long-term complica-tions do not require insulin to take up glucose. In these tissues, the intracellular level of glucosewill be higher than in the insulin-dependent ones.
198 14 Diabetes mellitus
before a meal, the time profile of insulin becoming available in the circulationapproximates the duration of the physiological postprandial peak.
The problem that remains then is to keep the level of insulin sufficientlyhigh in the periods between peaks. To this end, the rate of capillary uptakemust be slowed down even further. Preparations that have been conditioned fordelayed uptake are referred to as long-acting or “basal” insulins, as opposed tonative insulin and other short-acting “bolus” insulins. A combination of basaland bolus insulins, and optionally intermediate-acting ones, can then be usedto mimic the overall physiological insulin secretion profile. The rate of insulinmonomer release is influenced by a multitude of effects, which can be exploitedin the preparation of long- and intermediate-acting insulins:
1. Insulin is negatively charged at neutral pH, which will cause electrostaticmutual repulsion of the monomers, promoting dissociation. Adjusting the pHto lower values will reduce repulsion and promote aggregation.
2. Positively charged additives such as zinc and, more strongly, protamine—a small, positively charged nuclear protein obtained from the testes of rainbowtrout—will associate with insulin and promote its aggregation.
3. Insulin crystals dissolve more slowly than amorphous aggregates.
4. Addition of two positively charged arginine residues to the carboxy-ter-minus of the B chain, in combination with substitution of residue asparagineA21 by glycine, yields “glargine”, a derivative with stable, slow release kinetics.
5. Covalent linkage of a fatty acyl residue to lysine 29 yields “insulin de-temir”, which may form micellar aggregates or bind to other proteins, causingslower uptake into and slower clearance from the blood.
The exact mixture and dosage of slow- and fast-acting insulins has to beadjusted empirically with each patient. Traditionally, when developing an indi-vidual treatment plan, emphasis was placed on minimizing the number of dailyinsulin injections. One limitation of this approach is that the patient needs tocarefully synchronize his meals with his insulin application schedule. Moreover,the blood glucose level will often not be as tightly controlled as is desirable inorder to minimize the induction of diabetic long-term complications.
Tighter glucose control is the purpose of intensive insulin therapy, in whichfrequent measurements of blood glucose are used to guide the likewise morefrequent applications of insulin. One risk inherent in this approach is thathypoglycemia may result when a dose of insulin is applied before the previousones have been fully taken up into the circulation. In order to minimize thisrisk, it is desirable to accelerate the capillary uptake beyond the rate achievablewith native insulin. Several mutant insulins have been created that aggregateless readily than wild type insulin and therefore undergo faster capillary up-take. Insuline lispro, in which amino acid residues proline B28 and lysine B29are switched, and insulin aspart, which contains a mutant aspartate residueat position B28, are in clinical use and reportedly offer a reduced risk of hy-
14.9 Glucose assays 199
Hexamer Dimer Monomer
Capillary wall
Figure 14.5 Aggregation and capillary wall penetration of insulin. Zinc ions and highinsulin concentration promote the formation of hexamers, which don’t permeate acrossthe capillary walls. The aggregation equilibrium can be shifted either way by variouspoint mutations, which is exploited in the preparation of both fast- and slow-actinginsulins (see text for details).
poglycemia. Figure 14.6 illustrates how these changes reduce the stability ofinsulin aggregates.
The idea of just-in-time application of insulin leads logically to insulinpumps, which can release insulin continuously, much like the pancreatic islets.Ideally, the flow rate would be automatically controlled without any requireduser intervention by continuous measurement of the blood glucose level. Toavoid undulations in the feedback loop, the delay between the subcutaneous re-lease by the pump and the availability of insulin in the circulation should be assmall as possible; therefore, insulin preparations with minimized aggregationwill again be preferable.
Other insulin delivery methods have also been developed. Inhalable insulinwas available on the market for a short while. However, due to the concernsabout the long term effects of insulin on lungs and the accuracy in the dosage,demand was lower than expected, and the product was terminated.
14.9 Glucose assays
The dosage of insulin applied has to be adjusted according to the prevailingglucose concentration. This can be measured with a variety of enzymatic assays.One of these is the glucose oxidase/peroxidase assay:
glucose+O2 ---------------------------------------→ gluconate+H2O2 glucose oxidase
H2O2 + colorless pre-dye ---------------------------------------→ H2O+ dye peroxidase
In this coupled assay, the amount of dye formed in the second step is propor-tional to the glucose consumed in the first step. (Note that glucose oxidase isisolated from microbes but does not have a role in human metabolism).
200 14 Diabetes mellitus
Pro28
Glu21
Val3
Val12
Tyr16
Insulin dimer 1
Insulin dimer 2
Insulin dimer 3
Figure 14.6 Structure of the insulin hexamer. The hexamer is composed of threedimers and stabilized by two centrally placed zinc ions. The two monomers of eachdimer are highlighted in dark and light shades, respectively. In each dimer, the prolineresidue at position B29 of one monomer interacts with a patch of hydrophobic residues(valine B3, valine B12 and tyrosine B16) of the other monomer. In insulin lispro, prolineB28 is replaced with lysine, which destabilizes the interaction of the two monomers. Ininsuline aspart, aspartic acid replaces proline B28, which also breaks the hydrophobicinteraction and additionally creates electrostatic repulsion with glutamate B21 of theother monomer.
As a long-term parameter of diabetes adjustment, a parameter known asHbA1c is commonly used. HbA1 is one of the chains of hemoglobin. Its aminoterminus may undergo spontaneous, non-enzymatic reaction with glucose,
14.10 Diabetes type II 201
forming a Schiff base, which is called HbA1c:
Glucose−CH−−O+H2N−hemoglobin ---------------------------------------→ Glucose−CH−−N−hemoglobin+H2O
Since hemoglobin has a long lifetime, the percentage of HbA1 converted toHbA1c reflects the average concentration of glucose over a time span of severalweeks.
As stated above, spontaneous, non-enzymatic glucosylation of proteins isalso believed to contribute to the causation of long-term complications of di-abetes. Another potential mechanism is the lack of C-peptide. This moleculeused to be regarded only as a byproduct of insulin synthesis (Figure 13.2), andit is not present in pharmaceutical preparations of insulin. It has been reported,however, that treatment of diabetics with both insuline and C-peptid may re-duce the severity of diabetic complications such as degeneration of kidneys andperipheral nerves. Additional data from cell biological and animal experimentssupport the idea that C-peptide is a mediator in its own right. No receptor forC-peptide has been identified, however, and the effects in both experimentalanimals and in patients are moderate at best. It remains to be seen whetherand when C-peptide will become part of long-term therapy in type I diabeticpatients.
14.10 Diabetes type II
In diabetes type II, there is no destruction of the pancreatic islet cells, and thesecretion of insulin is not necessarily reduced but may be in the normal rangeor even enhanced. However, the peripheral tissues are less sensitive to insulin,and so increased amounts of insulin are required to achieve the necessary effecton the peripheral cells. The pathogenesis of diabetes type II is still not clearlyunderstood, so we will skip this scientific question and only note that diabetestype II is typically associated with overweight, and is most common in elderlypatients. The intracellular metabolic dysregulation is similar to that in diabetestype I, but typically less acute, since the residual insulin retains at least partialeffectiveness. Treatment of diabetes type II involves (1) diet to reduce weight;often sufficient in relatively young patients, but in the long run typically not;(2) sulfonylurea drugs such as tolbutamide, which will increase the endogenousinsulin secretion (Figure 13.3), (3) insulin therapy, just as in diabetes type I.
14.11 Diabetic coma and comatose diabetics
Untreated type I diabetes may first become manifest as diabetic coma, as statedabove; this condition is not common with untreated type II diabetes. However,with both forms, coma may arise from an overdose of insulin, which will leadto hypoglycemia, that is a lack of glucose in the blood. Since the brain depends
202 14 Diabetes mellitus
on glucose, it will pass out. Now, if you find a known diabetic in a comatosestate, what do you do – give him glucose or insulin? Since the hypoglycemiccoma is more immediately life-threatening than the hyperglycemic one, youmust always use glucose first. Only if that doesn’t help should you try insulin.3
14.12 Other forms of diabetes
While type I and II diabetes are the most common forms, diabetes may alsotake the following forms:
1. Secondary diabetes may occur due to other endocrine diseases. Exam-ples are diabetes due to cortisone- or epinephrine-producing tumors of theadrenal glands; both epinephrine and cortisone have metabolic effects that areantagonistic to insulin. Treatment consists, if possible, in curing the underlyingprimary disease.
2. Drug-induced diabetes. This is most commonly observed with cortisoneand other corticosteroids, which are used to treat chronical inflammatory orautoimmune diseases that are refractory to milder treatment. Increased bloodglucose is just one of their side effects.
14.13 The End
This concludes our overview of metabolism. If you made it this far, I hopethat you found at least some of it interesting. Please let me know about anynecessary corrections or desirable improvements to these notes. Thank you,and, if you are student in my class, good luck for your exam.
3This scenario is probably quite hypothetical nowadays, since fast and accurate glucose metersare now widely available. However, it used to be much more realistic in the days when glucoseassays could only be done in the lab.
