Carbohydrate Metabolism

Lai-Chu Wu, D. Phil.Department of Molecular and Cellular Biochemistry

laichu.wu@osumc.edu

1

Lecture Outline• Part One Digestion of carbohydrates and glycolysis.

The pathway, key enzymes, energy, and regulation.

• Part Two Gluconeogenesis. The key steps and enzymes between pyruvate and glucose, Cori cycle and the regulation of glycolysis and gluconeogenesis.

• Part three Glycogen metabolism. Important facts about glycogen, its structure, synthesis and degradation.

Part I

Carbohydrate Metabolism and

Glycolysis

Objectives

• Know the general structure of glucose, describe the pathway of glycolysis and the general chemical structures (that is functional groups) involved.

• Understand how glycolysis requires an initial investment of energy, but results in a net gain of energy.

• Explain how a molecule of glucose can produce two molecules of pyruvate.

• Give how availability of oxygen determines the conversion of pyruvate into lactic acid or acetyl coenzyme A.

• Know how glycolysis is regulated.

Carbohydrates: Introduction• Carbohydrates are the most abundant organic

molecule in nature.• Major sources of dietary carbohydrates

– Starch, from ingested plant cells– Glycogen, major form of carbohydrate storage in animals– Lactose (disaccharide of glucose and galactose) in milk and

fructose & sucrose in fruits

• Function:– Provide energy for our body– Give structure to cell walls and cell membranes– Serve as metabolic intermediates to amino acids and lipids– Components of nucleotides

Major pathways in carbohydrate metabolism

1. When energy is needed, glucose proceeds through glycolysis to pyruvate, which yields a small amount of energy

2. Pyruvate can be converted to acetyl-coenzyme A, enters the citric acid cycle and yields a large amount of energy.

3. Under anaerobic conditions, pyruvate is converted into lactate

Citric acid cycle acetyl-coenzyme A

Major pathways in carbohydrate metabolism continued

3. Glycogen, the storage form of glucose in animals, is synthesized by glycogenesis when glucose levels are high and degraded by glycogenolysis when glucose is in short supply.

4. Glucose can also be synthesized from noncarbohydrate precursors (amino acids from proteins and glycerol from lipids) by gluconeogenesis.

5. Glucose can also enter the pentose phosphate pathway, which yields NADPH (for reductive biosynthesis) and ribose 5-phosphate (for synthesis of nucleic acids).

Citric acid cycle acetyl-coenzyme A

Digestion of CarbohydratesCarbohydrate (CHO) digestion begins in the

mouth where salivary α-amylase breaks down polysaccharides into smaller polysaccharides and disaccharides.

Limited CHO digestion occurs in the stomach as α-amylase is inactivated by stomach acid.

Continues in the small intestine where pancreatic α-amylase together with maltase, sucrase and lactase released from the mucous lining hydrolyzes polysaccharides into monosaccharides (mainly glucose, fructose and galactose), which enter the bloodstream and transport to the cells.

Glycolysis - Overview• Glycolysis, an ancient and linear metabolic pathway, is found

in almost all organisms and occurs in the cytosol of every cell in our body.

• In glycolysis, a glucose molecule is split by a series of 10 enzymatic reactions into two molecules of pyruvate, and results in the net production of ATP, which fuels the body’s cells.

Glucose Activation

Conversion of glucose into glucose-6-phosphate

The glycolytic pathway

Step 1 – conversion of glucose to G6P

Glucose reacts with ATP to yield glucose 6-phosphate and ADP in a reaction catalyzed by hexokinase.

You do not need to memorize all the structures. I will highlight the functional groups involved for you to follow the reactions.

Hexokinase versus Glucokinase• There are four mammalian hexokinase isoforms that

have different enzyme kinetics with respect to different substrates and conditions, and functions

• Some isoforms have high affinity to glucose allowing cells such as brain and muscle to obtain glucose even at low glucose concentrations

• Glucokinase displays positive cooperativity with glucose, and functions when glucose concentration is high.

Hexokinase• In most tissues, the

phosphorylation of glucose to G6P is catalyzed by hexokinase, one of the three regulatory enzymes in glycolysis

• inhibited by its product G6P • Has a low Km (0.1 mM, i.e.

high affinity) for glucose, thus it is operating at Vmax under physiological blood glucose of ~5 mM

• Activity does not change with blood glucose levels

Glucokinase• Glucokinase is an isoform of

hexokinase• In liver parenchymal cells and

islet cells of the pancreas, glucokinase is the predominant enzyme responsible for the phosphorylation of glucose.

• has a high Km of 10 mM and Vmax which enables it to handle the high concentration of glucose that is present in the portal venous blood after a meal

• Not inhibited by G6P, allowing continue removal and “trap” glucose from blood as G6P.

Step 2 – Isomerization of G6P to F6P

Glucose 6-phosphate (aldose/aldohexose) is converted to fructose 6-phosphate (ketose/ketohexose).

The result is conversion of the six-membered glucose ring to a five-membered ring with a CH2OH group, which prepares the molecule for addition of another phosphate group in the next step.

Aldehydealcohol

Alcoholketone

Structures of G6P to F6P

aldehyde

ketone Addition of PO4 to –OH in the next

step

Step 3 - Phosphorylation of F6P by phosphofructokinase

• Fructose 6-phosphate reacts with ATP to yield fructose 1,6-bisphosphate plus ADP. Phosphofructokinase (PFK), the enzyme for step 3, provides a major control point in glycolysis.

• When the cell is short of energy, [AMP/ADP] builds up and activates phosphofructokinase.

• When energy is in good supply, [ATP] and [citrate] build up and inhibit this enzyme.

Energy investment stage of glycolysis• Stage 1 of glycolysis is

sometime called the energy investment phase in which 2 ATPs are consumed.

• Can you recall what are the two reactions that take up an ATP? the name of the enzymes, and what is common in the names of these enzymes?

Stag

e 2

stag

e 1

Steps 4 and 5 – Cleavage and Isomerization

Step 4. The six-carbon chain of fructose 1,6- bisphosphate is cleaved between C3 and C4 into two three-carbon pieces by the enzyme aldolase.

Step 5. Dihydroxyacetone phosphate is isomerized by the enzyme triose phosphate isomerase to glyceraldehyde 3-phosphate.

45

Stage I of glycolysis - Recap

• Glucose is phosphorylated twice and cleaved to form two molecules of glyceraldhyde-3-phosphate (G3P)

• Two ATP are consumed, and hence stage 1 is called the “investment” phase

• G3P is a high energy molecule “trapped” inside cell, ready for stage 2 of glycolysis (the energy generating stage)

• Starting from G3P and through 5 steps generate pyruvate

• It is called the energy generating phase because 2 ATPs and a NADH/H+ are produced.

• Since one glucose generates two G3P, in all stage 2 generates 2 pyruvate, 4 ATP, and 2 NADH/H+

7

Step 6 -10, stage 2 and energy generating phase -

Overview

Step 6 - Oxidation & Phosphorylation by glyceraldehyde 3-phosphate dehydrogenase

• G3P is converted to 1,3BPG• Two reactions actually occur:

• aldehyde group in G3P is first oxidized to a carboxylic acid and

• phosphorylated by glyceraldehyde 3-phosphate dehydrogenase.

• The oxidation reaction (G3P to 1,3BPG) is coupled to the reduction of NAD+ to NADH/H+

• the source of phosphate is inorganic phosphate, but not ATP, so this phosphorylation step does not consume ATP

aldehyde

acid

Step 7 - Phosphorylation by Phosphoglycerate Kinase

• A phosphate group from 1,3-BPG is transferred to ADP, resulting in the synthesis of ATP.

• It is catalyzed by phosphoglycerate kinase.

• First energy releasing step in glycolysis

Step 8 - Isomerization by phosphoglycerate mutase

• A phosphate group is transferred from 3-Phosphoglycerate at carbon 3 to carbon 2, generating

2-Phosphoglycerate• The shift of the PO4

group from C3 to C2 by phosphoglycerate mutase is reversible.

• The carbon in COOH is labeled as carbon #1

3 2 1

Step 9 - Dehydration by Enolase

Dehydration reaction resulting in loss of water from 2-phosphoglycerate producing phosphoenolpyruvate (PEP), which contains a high energy enol phosphate group

Step 10 - Phosphorylation by Pyruvate Kinase

• Transfer of the phosphate group from PEP to ADP yields pyruvate and generates ATP

• Second energy releasing step in glycolysis

Energy Production In Glycolysis

7

• Starting from glucose, glycolysis uses 2 ATP and 2 NAD, and produces 4 ATP and 2 NADH.

• ATP is consumed in the reactions catalyzed by hexokinase (step 1) and phosphofructokinase (step 3).

• ATP is produced in the reactions catalyzed by phosphoglycerate kinase (step 7) and pyruvate kinase (step 10).

• NAD is converted to NADH in the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase (step 6).

Stag

e 2

stag

e 1

The Fate of Pyruvate

• The fate of pyruvate depend on metabolic conditions.

pyruvate

glycolysis

lactate

acetyl-SCoA

Anaerobiccondition

Tricarboxylic acid cycle

lipid metabolismProtein metabolism

aerobiccondition

• Fructose is converted to glycolysis intermediates in 2 ways:– In muscle cells, it is phosphorylated to F6P and enters in

step 3 of glycolysis. – In the liver, it is converted to G3P and enters in step 6.

• Galactose is converted to G6P by a five-step pathway

Entry of Other Sugars into Glycolysis

A couple of fun facts about the final step of glycolysis

• In the final step, step 10, of glycolysis, PEP is converted into pyruvate by pyruvate kinase. In this reaction, a PO4 is removes, so the enzyme was actually named for the reversed reaction, which does not exist

• Have you ever wonder why you got hot when you run? Step 10 releases lots of energy, almost enough to generate 2 ATP. Only one ATP is produced and the remaining energy is lost as heat.

Carbohydrate Metabolism Part 1 Quiz

Part II

Gluconeogenesis

Objectives

• Know the gluconeogenesis pathway• Evaluate the pathway and key steps of

glycolysis and gluconeogenesis to appreciate their reciprocal regulation

• Know how pyruvate, lactate and alanline enter gluconeogenesis

Gluconeogenesis - Introduction• The anabolic counterpart to glycolysis is gluconeogenesis,

which occurs mainly in the liver (~90%) and kidney (~10%).

• Gluconeogenesis is the synthesis of glucose typically from 3-carbon precursors such as lactate, pyruvate, alanine and glycerol formed by metabolism in the peripheral tissues.

• The purpose of this pathway is to provide the body with glucose under physiological conditions when storage of glucose (in the form of glycogen) is depleted and there is no glucose available from the gut.

• This pathway becomes critical during the early stages of starvation and fasting. Failure of gluconeogenesis is usually fatal.

Substrates for gluconeogenesis

• Pyruvate (not from glycolysis)• Lactate, glucose-lactate cycle (AKA Cori

cycle, discovered by Carl and Gertrude Cori)

• Amino acids (glucose-alanine cycle)• Glycerol

• The anaerobic metabolism of glucose by red blood cells, skeletal muscle and other peripheral tissues leads to the formation of lactate.

• The reaction involves the reduction of the ketone group at C2 of pyruvate to an alcohol group forming lactate

• In this reaction, NADH/H+ is oxidized to NAD+

• This reaction is important because it replenishes the NAD+ used in step 6 of glycolysis. Without NAD+, glycolysis will stop.

Anaerobic metabolism of glucose forms lactate

Lactate as a substrate for gluconeogenesis: Glucose-lactate (Cori) cycle

1. Lactate produced in muscle under anaerobic conditions and carried by blood is converted to glucose in the liver.

2. The liver releases glucose, which is carried by the blood to the skeletal muscle.

3. Cori cycle is the shuttling of glucose from the liver to skeletal muscle and of lactate from skeletal muscle back to the liver, where it is resynthesized to glucose.

Alanine as a substrate for gluconeogenesis: The glucose- alanine cycle

• During starvation, skeletal muscle protein are degraded to yield amino acids and then ammonia and glutamate.

• The transamination of pyruvate with glutamate yields alanine which is transported through the blood to the liver.

Glucose- alanine cycle (continued)

• In the liver, transamination reforms pyruvate and glutamate. • Pyruvate is converted to glucose, which can be transported back

to the skeletal muscle and used in glycolysis to yield ATP.• Function:

• Provide carbon as a precursor of glucose synthesis in the liver. • Transport nitrogen atoms to the liver for excretion as urea.

Glycerol as a substrate for gluconeogenesis

• Glycerol is formed in the adipose tissues by lipolysis of triacylglycerol when metabolic fuel is scarce

• Glycerol is delivered to the liver through blood• In the liver, glycerol is phosphorylated to form glycerol-3-

phosphate, an intermediate of glycolysis (step 7).

adipocyte liver

triacylglycerol glycerol

Fatty acids

Glycerol-3-P G3PDHAP

glucose

The pathway of gluconeogenesis largely is the reversed reaction of glycolysis

• The gluconeogenesis pathway starts with pyruvate, ends with glucose, and consists of 11 reactions.

• Of the 11 reactions in gluconeogenesis, 7 are catalyzed by the same enzymes used as in glycolysis, but for the reversed reactions

• three steps in glycolysis releases are highly exergonic (release lots of energy) and therefore are not reversible.

The three irreversible steps in glycolysis

• Three steps in glycolysis are not reversed by gluconeogenesis:

(1) glucose to glucose 6-phosphate

(2) fructose 6-phosphate to fructose 1,6-bisphosphate

(3) phosphoenolpyruvate to pyruvate

Enzymes specific for gluconeogenesis or glycolysis

Substrate/product glycolysis gluconeogenesis

Glucose to G6P hexokinase/glucokinase (GK)

Glucose-6-phosphatase (G6Pase)

F6P to F1,6BP Phosphofructose kinase (6PF-1K or PFK)

fructose-1,6-bisphosphatase

PEP to pyruvate Pyruvate kinase (PK) NIL

Pyruvate to oxaloacetate (OAA)

NIL pyruvateCarboxylase (PC)

OAA to PEP NIL phosphoenolpyruvate carboxykinase(PEPCK)

Note that conversion of PEP to pyruvate releases lots of energy and therefore two steps are needed to convert pyruvate back to PEP.

Reactions specific for gluconeogenesis

• Seven reactions in gluconeogenesis are the reversed reactions of glycolysis (will not be further discussed)

• Reactions to be discussed:1. Pyruvate to PEP2. F 1,6 BP to F6P3. G6P to glucose

Conversion of pyruvate into phosphoenolpyruvate

• Conversion of pyruvate to PEP involves two steps.

• Pyruvate is first converted to oxaloacetate. In this reaction, pyruvate, carbon dioxide and ATP are converted to oxaloacetate, ADP and inorganic phosphate

• Oxaloacetate is then converted to PEP, using GTP

• one ATP and one GTP is used to form PEP from pyruvate. An equivalent of 2 ATP is used. Because 2 molecules of pyruvate make one molecule of glucose, a total of 4 ATP are used.

Conversion of F1,6-bisphosphate to fructose-6-phosphate

• Hydrolysis of F1,6BP by F1,6 phosphatase (F1,6P) to F6P bypasses the PFK reaction.

• This reaction is an important regulation in gluconeogenesis

• high ATP and low AMP (=energy rich state) stimulates F1,6P (low ATP & high AMP=energy poor state)

• F2,6BP inhibits F1,6P (F2,6BP activates PFK; hence reciprocal control of glucose synthesis and breakdown)

Conversion of glucose-6-phosphate to glucose

• Hydrolysis of G6P by glucose-6-phosphate bypasses the hexokinase reaction in glycolysis.

• Liver and kidney are the only organs contains the glucose-6-phosphatase

• Muscle lacks this enzyme, so G6P formed by gluconeogenesis can only be used by muscle.

Energy in Gluconeogenesis

• Gluconeogenesis is an endergonic reaction that requires energy.

• In gluconeogenesis, the conversion of two moles of pyruvate to 1 mole of glucose uses 4 moles of ATP and 2 moles of GTP

Carbohydrate Metabolism Part 2 Quiz

Part III

Glycogen metabolism

Objectives

• Know the general structure of glycogen and discuss the metabolic advantages of such structure

• Know how the glycogen molecule is synthesized and degraded

• Know the different ways by which energy is generated from glycogen in the liver and muscle

Structure of Glycogen

1

• Glycogen is a polymer of glucose (up to 120,000 glucose residues) and is a primary carbohydrate storage form in animals.

• The polymer is composed of units of glucose linked by alpha(1-4) glycosidic bonds in the linear molecule and alpha(1-6) glycosidic bonds at branch points.

• Liver and skeletal muscle are the primary sites in the body where glycogen is found

Glycogen as the storage of glucose

• The end of the molecule containing a free carbon number one on glucose is called a reducing end. The other ends are all called non-reducing ends.

• Because glycogen contains so many glucoses, it provides a quick source of glucose when needed and a place to store excess glucose when glucose concentrations in the blood rise.

• The branching of glycogen is an important feature of the molecule metabolically as well. Since glycogen is broken down from the "ends" of the molecule, more branches translate to more ends, and more glucose that can be released at once.

glucose

Synthesis of glycogen - glycogenesis

• Glycogenesis occurs when glucose concentrations are high.

• Glycogenesis involves 4 steps:1. Glucose is phosphorylated to

Glucose 6-Phosphate (G6P) 2. G6P is isomerized to G1P3. UDP is added to G1P to form

Glucose-UDP4. Glucose-UDP is added to the

growing glycogen chain

1

2

3

4

Glycogen synthesis

UDP-glucose + glycogen (n) glycogen (n+1) + UDP

Addition of one glucose to the glycogen chain

Non-reducing end

a-1,4-glycosidic bond

1 2 3 4 5 6

Formation of Branch chains in glycogen

• As glucose subunits are added to the ends of a glycogen chain, segments of the glycogen chain can be transferred onto the neighboring chain, forming a-1,6-linkage.

Glycogen degradation (Glycogenolysis)

glycogen phosphorylase

• Occurs in muscle cells when energy needed and in liver when blood glucose is low.

• Glycogen phosphorylase catalyzes glycogen phosphorylysis (bond cleavage by the substitution of a phosphate group) and yields glucose-1-phosphate (G1P)

Fate of glucose-1-phosphate

liver

muscle

Glycogen degradation - Debranching

• Phosphorylase removes glucose one at a time from the non-reducing end, but stops at a point four residues away from the branch.

• A transferase enzyme moves the outer three residues from a branch to of the adjacent branch, and leaves one at the branch point.

• Then a second enzyme, alpha-1,6-glucosidase, clips the alpha-1,6 bond, releasing the last residue of the stub and converting the chain into a linear polymer that phosphorylase can continue to act on.

Non-reducing end

Regulation of glycogen metabolism• Because of the importance

of maintaining blood glucose levels, the synthesis and degradation of glycogen are tightly regulated

• Under well fed state, G6P stimulates glycogen synthase, converting G1P into glycogen, whereas glucose, ATP and G6P inhibit glycogen phosphorylase, preventing the breakdown of glycogen

• Glycogen metabolism is also under allosteric controls and hormonal regulation.

Summary -Metabolic Pathways of Glucose

Pathway function

Glycolysis Converts glucose to pyruvate

gluconeogenesis Synthesis of glucose from noncarbohydrate sources (pyruvate, lactate, alanline and glycerol)

glycogenogenesis Synthesizes of glycogen from glucose

glycogenolysis Degradation of glycogen to G1P

Carbohydrate Metabolism Part 3 Quiz

References

• Principles of Medical Biochemistry, Meisenberg and Simmons, 3rd edition, chapter 22.

• Lippincott’s Illustrated Reviews: Biochemistry, Champe, Harvey, and Ferrier, Chapter 13

Thank you

• Thank you for completing this module.• If you have any questions or comments,

please contact me.• laichu.wu@osumc.edu