UNIT- II€¦ · Reaction 1: Formation of Citrate The first reaction of the cycle is the...

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JAIPUR COLLEGE OF PHARMACY, JAIPUR B.PHARMACY, FIRST YEAR, SECOND SEMESTER

BIOCHEMISTRY Prepared by: Dr. Rakesh Kumar Gupta

UNIT- II

Glycolysis

Glycolysis, which translates to "splitting sugars", is the process of releasing

energy within sugars. In glycolysis, a six-carbon sugar known as glucose is split

into two molecules of a three-carbon sugar called pyruvate. This multistep

process yields two ATP molecules containing free energy, two pyruvate

molecules, two high energy, electron-carrying molecules of NADH, and two

molecules of water.

Definition

Glycolysis can be defined as the sequence of reactions for the breakdown of

Glucose (6-carbon molecule) to two molecules of pyruvic acid (3-carbon

molecule) under aerobic conditions; or lactate under anaerobic conditions along

with the production of small amount of energy.

Glycolysis

Glycolysis is the process of breaking down glucose.

Glycolysis can take place with or without oxygen.

Glycolysis produces two molecules of pyruvate, two molecules of ATP,

two molecules of NADH, and two molecules of water.

Glycolysis takes place in the cytoplasm.

There are 10 enzymes involved in breaking down sugar. The 10 steps of

glycolysis are organized by the order in which specific enzymes act upon

the system.

https://www.thoughtco.com/pathway-most-atp-per-glucose-molecule-608200




Figure 1 Glycolysis Cycle




Glycolysis can occur with or without oxygen. In the presence of oxygen,

glycolysis is the first stage of cellular respiration. In the absence of oxygen,

glycolysis allows cells to make small amounts of ATP through a process of

fermentation.

Glycolysis takes place in the cytosol of the cell's cytoplasm. A net of two ATP

molecules are produced through glycolysis (two are used during the process and

four are produced). Following are the 10 steps of glycolysis:

Step 1

The enzyme hexokinase phosphorylates or adds a phosphate group to glucose

in a cell's cytoplasm. In the process, a phosphate group from ATP is transferred

to glucose producing glucose 6-phosphate or G6P. One molecule of ATP is

consumed during this phase.

Step 2

The enzyme phosphoglucomutase isomerizes G6P into its isomer fructose 6-

phosphate or F6P. Isomers have the same molecular formula as each other but

different atomic arrangements.

Step 3

The kinase phosphofructokinase uses another ATP molecule to transfer a

phosphate group to F6P in order to form fructose 1,6-bisphosphate or FBP. Two

ATP molecules have been used so far.




Step 4

The enzyme aldolase splits fructose 1,6-bisphosphate into a ketone and an

aldehyde molecule. These sugars, dihydroxyacetone phosphate (DHAP) and

glyceraldehyde 3-phosphate (GAP), are isomers of each other.

Step 5

The enzyme triose-phosphate isomerase rapidly converts DHAP into GAP

(these isomers can inter-convert). GAP is the substrate needed for the next step

of glycolysis.

Step 6

The enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves

two functions in this reaction. First, it dehydrogenates GAP by transferring one

of its hydrogen (H⁺) molecules to the oxidizing agent nicotinamide adenine

dinucleotide (NAD⁺) to form NADH + H⁺.

Next, GAPDH adds a phosphate from the cytosol to the oxidized GAP to form

1,3-bisphosphoglycerate (BPG). Both molecules of GAP produced in the

previous step undergo this process of dehydrogenation and phosphorylation.

Step 7

The enzyme phosphoglycerokinase transfers a phosphate from BPG to a

molecule of ADP to form ATP. This happens to each molecule of BPG. This

reaction yields two 3-phosphoglycerate (3 PGA) molecules and two ATP

molecules.




Step 8

The enzyme phosphoglyceromutase relocates the P of the two 3 PGA

molecules from the third to the second carbon to form two 2-phosphoglycerate

(2 PGA) molecules.

Step 9

The enzyme enolase removes a molecule of water from 2-phosphoglycerate to

form phosphoenolpyruvate (PEP). This happens for each molecule of 2 PGA

from Step 8.

Step 10

The enzyme pyruvate kinase transfers a P from PEP to ADP to form pyruvate

and ATP. This happens for each molecule of PEP. This reaction yields two

molecules of pyruvate and two ATP molecules.

Significance of the Glycolysis Pathway

1. Glycolysis is the only pathway that is taking place in all the cells of the

body.

2. Glycolysis is the only source of energy in erythrocytes.

3. In Strenuous exercise, when muscle tissues lack enough oxygen,

anaerobic glycolysis forms the major source of energy for muscles.

4. The Glycolytic pathway may be considered as the preliminary step before

complete oxidation.

5. The glycolytic pathway provides carbon skeletons for synthesis of non

essential amino acids as well as glycerol part of fat.

6. Most of the reactions of the glycolytic pathway are reversible, which are

also used for gluconeogenesis.

https://www.thoughtco.com/why-is-water-a-polar-molecule-609416




Energy Yield in Aerobic Glycolysis

Step Enzyme Source No. of ATP

1 Hexokinase – -1

3 Phosphofructokinase – -1

6 Glyceraldehyde-3- phosphate

dehydrogenase

NADH (+3) x 2 = +6

7 Phosphoglycerate kinase ATP (+1) x 2 = +2

10 Pyruvate kinase ATP (+1) x 2 = +2

Net

Yield

8 ATPs




Krebs (Citric Acid) Cycle

It is also known as Tri Carboxylic Acid (TCA) cycle. In prokaryotic cells, the

citric acid cycle occurs in the cytoplasm; in eukaryotic cells, the citric acid cycle

takes place in the matrix of the mitochondria.

The cycle was first elucidated by scientist “Sir Hans Adolf Krebs” (1900 to

1981). He shared the Nobel Prize for physiology and Medicine in 1953 with

Fritz Albert Lipmann, the father of ATP cycle.

Figure 2 Citric Acid Cycle




The process oxidises glucose derivatives, fatty acids and amino acids to carbon

dioxide (CO2) through a series of enzyme controlled steps. The purpose of the

Krebs Cycle is to collect (eight) high-energy electrons from these fuels by

oxidising them, which are transported by activated carriers NADH and FADH2

to the electron transport chain. The Krebs Cycle is also the source for the

precursors of many other molecules, and is therefore an amphibolic pathway

(meaning it is both anabolic and catabolic).

acetyl CoA + 3 NAD + FAD + ADP + HPO4-2 —————> 2 CO2 + CoA + 3

NADH+ + FADH+ + ATP

Reaction 1: Formation of Citrate

The first reaction of the cycle is the condensation of acetyl-

CoA with oxaloacetate to form citrate, catalyzed by citrate synthase.

Once oxaloacetate is joined with acetyl-CoA, a water molecule attacks the

acetyl leading to the release of coenzyme A from the complex.




Reaction 2: Formation of Isocitrate

The citrate is rearranged to form an isomeric form, isocitrate by an

enzyme acontinase.

In this reaction, a water molecule is removed from the citric acid and then put

back on in another location. The overall effect of this conversion is that the –

OH group is moved from the 3′ to the 4′ position on the molecule. This

transformation yields the molecule isocitrate.

Reaction 3: Oxidation of Isocitrate to α-Ketoglutarate

In this step, isocitrate dehydrogenase catalyzes oxidative decarboxylation

of isocitrate to form α-ketoglutarate.

In the reaction, generation of NADH from NAD is seen. The enzyme isocitrate

dehydrogenase catalyzes the oxidation of the –OH group at the 4′ position of

isocitrate to yield an intermediate which then has a carbon dioxide molecule

removed from it to yield alpha-ketoglutarate.




Reaction 4: Oxidation of α-Ketoglutarate to Succinyl-CoA

Alpha-ketoglutarate is oxidized, carbon dioxide is removed, and coenzyme A

is added to form the 4-carbon compound succinyl-CoA.

During this oxidation, NAD+ is reduced to NADH + H+. The enzyme that

catalyzes this reaction is alpha-ketoglutarate dehydrogenase.




Reaction 5: Conversion of Succinyl-CoA to Succinate

CoA is removed from succinyl-CoA to produce succinate.

The energy released is used to make guanosine triphosphate (GTP) from

guanosine diphosphate (GDP) and Pi by substrate-level phosphorylation. GTP

can then be used to make ATP. The enzyme succinyl-CoA synthase catalyzes

this reaction of the citric acid cycle.

Reaction 6: Oxidation of Succinate to Fumarate

Succinate is oxidized to fumarate.

During this oxidation, FAD is reduced to FADH2. The enzyme succinate

dehydrogenase catalyzes the removal of two hydrogens from succinate.




Reaction 7: Hydration of Fumarate to Malate

The reversible hydration of fumarate to L-malate is catalyzed by fumarase

(fumarate hydratase).

Fumarase continues the rearrangement process by adding Hydrogen and

Oxygen back into the substrate that had been previously removed.




Reaction 8: Oxidation of Malate to Oxaloacetate

Malate is oxidized to produce oxaloacetate, the starting compound of the citric

acid cycle by malate dehydrogenase. During this oxidation, NAD+ is reduced

to NADH + H+.

ATP Generation

Total ATP = 12 ATP

3 NAD+ = 9 ATP

1 FAD = 2 ATP

1 ATP = 1 ATP

Reviewing the whole process, the Krebs cycle primarily transforms the acetyl

group and water, into carbon dioxide and energized forms of the other reactants.




Significance of Krebs Cycle

1. Intermediate compounds formed during Krebs cycle are used for the

synthesis of biomolecules like amino acids, nucleotides, chlorophyll,

cytochromes and fats etc.

2. Intermediate like succinyl CoA takes part in the formation of chlorophyll.

3. Amino Acids are formed from α- Ketoglutaric acid, pyruvic acids and

oxaloacetic acid.

4. Krebs cycle (citric Acid cycle) releases plenty of energy (ATP) required

for various metabolic activities of cell.

By this cycle, carbon skeleton are got, which are used in process of growth and

for maintaining the cells.




Pentose phosphate pathway (PPP) or Hexose mono-phosphate

(HMP) shunt

The hexose monophosphate shunt, also known as the pentose phosphate

pathway, is a unique pathway used to create products essential in the body for

many reasons. The HMP shunt is an alternative pathway to glycolysis and is

used to produce ribose-5-phosphate and nicotinamide adenine dinucleotide

phosphate (NADPH). This pathway occurs in the oxidative and non-oxidative

phases, each comprising a series of reactions. The HMP shunt also has

significance in the medical world, as enzyme or co-factor deficiencies can have

potentially fatal implications on the affected patients.

• Pentose phosphate pathway is an alternative pathway to glycolysis and

TCA cycle for oxidation of glucose.

• It is a shunt of glycolysis

• It is also known as hexose monophosphate (HMP) shunt or

phosphogluconate pathway.

• It occurs in cytoplasm of both prokaryotes and eukaryotes

• Pentose phosphate pathway starts with glucose and it is a multi-steps

reaction.

The sequence of reactions are divided into two types.

I) oxidative reaction phase

II) Non-oxidative reaction phase




Figure 3 HMP Shunt




Oxidative phase

Figure 4 Oxidative Phase

First four reactions are irreversible and oxidative in which glucose molecule is

oxidized twice generating two molecules of NADPH and glucose is converted

into Ribose-5 phosphate.

1st step: conversion of glucose to glucose-6 phosphate.

This reaction is catalyzed by the enzyme hexokinase and a molecule of

ATP is utilized. This reaction is actually a primary step of glycolysis.

2nd step: conversion of glucose-6 phosphate to 6-phosphogluconolactone.

This reaction is catalyzed by an enzyme glucose-6 phosphate

dehydrogenase (G6PD) in the presence of Mg++ ion.




In this reaction a molecule of NADPH is produced.

3rd step: conversion of 6-phosphogluconolactone to 6-phosphogluconate

This reaction is a hydrolysis reaction catalyzed by hydrolase enzyme

4th step: conversion of 6-phosphogluconate to ribose-5 phosphate

This reaction is catalyzed by the enzyme 6-phosphogluconate

dehydrogenase to produce 3-keto-6-phosphogluconate which undergoes

decarboxylation to produce ribulose-5 phosphate.

In this reaction a molecule of NADPH is generated.

Non oxidative phase




Oxidative reactions are followed by a series reversible sugar phosphate

inter-conversion reaction.

Ribulose-5-phosphate is epimerized to produce xylulose 5-phosphate in

the presence of enzyme phosphor pentose epimerase. Similarly ribulose-

5-phosphate is also keto-isomerized into ribose 5-phosphate.

Xylulose-5-phsphate transfer two carbon moiety to ribose 5-phospahate

in the presence of enzyme transketolase to form sedoheptulose-7-

phosphate and glyceraldehyde 3—phosphate.

Sedoheptulose -7-phosphate transfer three carbon moiety to

glyceraldehyde -3-phosphate to form fructose 6-

phopsphate and erythrose 4-phosphate in the presence of enzyme

transaldolase.

Transketolase enzyme catalyse the transfer of two carbon moiety from

Xylulose-5-phsphate to erythrose-4- phosphate to form fructose-6-

phosphate and glyceraldehyde-3-phosphate.

Fructose-6-phosphate and glyceraldehyde-3-phosphate is later enter into

glycolysis and kreb’s cycle.

The rate and direction of reversible reaction depends upon the needs of

cell.

If cell needs only NADPH then fructose-phosphate and glyceraldehyde-3-

phosphate are converted back to glucose by reverse glycolysis, otherwise

converted to pyruvate and enter TCA cycle generating ATPs.

Significance of Pentose phosphate pathway

HMP is only the cytoplasmic pathway that generates NADPH

NADPH is produced in this pathway acts as reducing agent during

biosynthesis of various molecules eg. fattyacids.




This pathway generates 3, 4, 5, 6 and 7 carbon compounds which are

precursors for biosynthesis of other molecules. Eg nucleotides are

synthesized from ribose-5-phsophate.

Pentose phosphate pathway is very essential for cell lacking

mitochondria (eg.RBCs) for generation of NADPH.

Triose, tetrose, pentose, hexose and heptose sugar are generated as

intermediate products in pentose phosphate pathway.

NADPH is also used to reduce (detoxify) Hydrogen peroxide in cell.

Resistance to malaria in some Africans are associated with deficiency of

glucose-6-phosphate dehydrogenase enzyme because malarial parasites

depend upon HMP shunt to reduce glutathione in RBCs.




Glycogen Metabolism

Glycogen is a readily mobilized storage form of glucose. It is a very large,

branched polymer of glucose residue that can be broken down to yield glucose

molecules when energy is needed. Most of the glucose residues in glycogen are

linked by α-1,4-glycosidic bonds. Branches at about every tenth residue are

created by α-1,6-glycosidic bonds. Recall that α-glycosidic linkages form open

helical polymers, whereas β linkages produce nearly straight strands that form

structural fibrils, as in cellulose

There are 6 major steps are involved in the Glycogenolysis:

Step 1: Glucose Phosphorylation

Glucose is phosphorylated into Glucose-6-Phosphate, a reaction that is common

to the first reaction in the pathway of glycolysis from Glucose.

This reaction is catalyzed by Hexokinase in Muscle and Glucokinase in the

Liver.




Step 2: Glc-6-P To Glc-1-P Conversion

Glucose-6-P is converted to Glc-1-Phosphate in a reaction catalyzed by the

enzyme “Phosphoglucomutase”.

Glucose-6-P + Enz-P <—> Glucose-1,6-bis Phosphate + Enz <—

> Glucose-1-Phosphate + Enzyme-P

Step 3: Attachment of UTP to Glc-1-P

Glucose-1-P reacts with Uridine triphosphate (UTP) to form the active

nucleotide Uridine diphosphate Glucose (UDP-Glc). The reaction is catalyzed

by the enzyme “UDPGlc Pyrophosphorylase”.

UTP+ Glucose-1-P <-> UDPGlc+ PPi

Step 4: Attachment of UDP-Glc to Glycogen Primer

A small fragment of pre-existing glycogen must act as a “Primer” (also called

GLYCOGENIN) to initiate glycogen synthesis. The Glycogenin can accept

glucose from UDP-Glc.

The hydroxyl group of the amino acid tyrosine of Glycogenin is the site at

which the initial glucose unit is attached. The enzyme Glycogen initiator

synthase transfers the first molecule of Glucose to Glycogenin. Then glycogenin

itself takes up a for glucose residues to form a fragment of primer which serves

as an acceptor for the rest of the glucose molecules.

Step 5: Glycogen Synthesis by Glycogen Synthase




Glycogen synthase, the enzyme transfers the Glucose from UDP-Glc to the non-

reducing end of Glycogen to form alpha 1,4-linkages.

Glycogen synthase catalyzes the synthesis of a linear unbranched molecule with

alpha-1,4-glycosidic linkages.

Step 6: Glycogen Branches Formation

In this step, the formation of branches is brought about by the action of a

branching enzyme, namely branching enzyme (amylo-[1—>4]—>[1—>6]-

transglucosidase).

This enzyme transfers a small fragment of five to eight glucose residues from

the non-reducing end of the glycogen chain to another glucose residue where it

is linked by the alpha-1,6 bond.

It leads to the formation of a new non-reducing end, besides the existing one.

The glycogen chain will be elongated and branched.

The overall reaction of Glycogenesis,

(Glucose)n + Glucose +2 ATP -> (Glucose)n+1 +2 ADP + Pi

Two ATP molecules will utilize in this process. One is required for

the phosphorylation of Glucose and the other is needed for conversion of UDP

to UTP.




GLYCOGEN STORAGE DISEASE

A glycogen storage disease (GSD, also glycogenosis and dextrinosis) is

a metabolic disorder caused by enzyme deficiencies affecting

either glycogen synthesis, glycogen breakdown or glycol sis (glucose

breakdown), typically in muscles and/or liver cells.

GSD has two classes of cause: genetic and acquired.

Genetic GSD is caused by any inborn error of metabolism (genetically

defective enzymes) involved in these processes. In livestock, acquired GSD is

caused by intoxication with the alkaloid castanospermine.

Glycogen storage disease type I (also known as GSDI or von Gierke disease) is

an inherited disorder caused by the buildup of a complex sugar

called glycogen in the body's cells. The accumulation of glycogen in certain

organs and tissues, especially the liver, kidneys, and small intestines, impairs

their ability to function normally.

Signs and symptoms of this condition typically appear around the age of 3 or 4

months, when babies start to sleep through the night and do not eat as frequently

as newborns. Affected infants may have low blood sugar (hypoglycemia),

which can lead to seizures. They can also have a buildup of lactic acid in the

body (lactic acidosis), high blood levels of a waste product called uric acid

(hyperuricemia), and excess amounts of fats in the blood (hyperlipidemia). As

they get older, children with GSDI have thin arms and legs and short stature. An

enlarged liver may give the appearance of a protruding abdomen. The kidneys

may also be enlarged. Affected individuals may also have diarrhea and deposits

of cholesterol in the skin (xanthomas).

https://ghr.nlm.nih.gov/art/large/glucose-glycogen.jpeg




People with GSDI may experience delayed puberty. Beginning in young to mid-

adulthood, affected individuals may have thinning of the bones (osteoporosis), a

form of arthritis resulting from uric acid crystals in the joints (gout), kidney

disease, and high blood pressure in the blood vessels that supply the lungs

(pulmonary hypertension). Females with this condition may also have abnormal

development of the ovaries (polycystic ovaries). In affected teens and adults,

tumors called adenomas may form in the liver. Adenomas are usually

noncancerous (benign), but occasionally these tumors can become cancerous

(malignant).

Researchers have described two types of GSDI, which differ in their signs and

symptoms and genetic cause. These types are known as glycogen storage

disease type Ia (GSDIa) and glycogen storage disease type Ib (GSDIb). Two

other forms of GSDI have been described, and they were originally named types

Ic and Id. However, these types are now known to be variations of GSDIb; for

this reason, GSDIb is sometimes called GSD type I non-a.

Many people with GSDIb have a shortage of white blood cells (neutropenia),

which can make them prone to recurrent bacterial infections. Neutropenia is

usually apparent by age 1. Many affected individuals also have inflammation of

the intestinal walls (inflammatory bowel disease). People with GSDIb may have

oral problems including cavities, inflammation of the gums (gingivitis), chronic

gum (periodontal) disease, abnormal tooth development, and open sores (ulcers)

in the mouth. The neutropenia and oral problems are specific to people with

GSDIb and are typically not seen in people with GSDIa.

https://ghr.nlm.nih.gov/art/large/bone-with-osteoporosis.jpeg

https://ghr.nlm.nih.gov/art/large/cancer.jpeg




Gluconeogenesis

Gluconeogenesis (GNG) is a metabolic pathway that results in the generation

of glucose from certain non-carbohydrate carbon substrates. From breakdown

of proteins, these substrates include glucogenic amino acids (although

not ketogenic amino acids); from breakdown of lipids (such as triglycerides),

they include glycerol, odd-chain fatty acids (although not even-chain fatty acids,

see below); and from other steps in metabolism they

include pyruvate and lactate. Although most gluconeogenesis occurs in the

liver, the relative contribution of gluconeogenesis by the kidney is increased in

diabetes and prolonged fasting.

Gluconeogenesis is one of several main mechanisms used by humans and many

other animals to maintain blood glucose levels, avoiding low levels

(hypoglycemia). Other means include the degradation

of glycogen (glycogenolysis) and fatty acid catabolism.

Gluconeogenesis is a ubiquitous process, present in plants, animals, fungi,

bacteria, and other microorganisms. In vertebrates, gluconeogenesis takes place

mainly in the liver and, to a lesser extent, in the cortex of the kidneys.

In ruminants, this tends to be a continuous process. In many other animals, the

process occurs during periods of fasting, starvation, low-carbohydrate diets, or

intense exercise. The process is highly endergonic until it is coupled to the

hydrolysis of ATP or GTP, effectively making the process exergonic. For

example, the pathway leading from pyruvate to glucose-6-phosphate requires 4

molecules of ATP and 2 molecules of GTP to proceed spontaneously.

Gluconeogenesis is often associated with ketosis.

https://en.wikipedia.org/wiki/Metabolic_pathway

https://en.wikipedia.org/wiki/Glucose

https://en.wikipedia.org/wiki/Carbohydrate

https://en.wikipedia.org/wiki/Protein

https://en.wikipedia.org/wiki/Glucogenic_amino_acid

https://en.wikipedia.org/wiki/Ketogenic_amino_acid

https://en.wikipedia.org/wiki/Lipid

https://en.wikipedia.org/wiki/Triglyceride

https://en.wikipedia.org/wiki/Glycerol

https://en.wikipedia.org/wiki/Metabolism

https://en.wikipedia.org/wiki/Pyruvic_acid

https://en.wikipedia.org/wiki/Lactic_acid

https://en.wikipedia.org/wiki/Blood_sugar

https://en.wikipedia.org/wiki/Hypoglycemia

https://en.wikipedia.org/wiki/Glycogen

https://en.wikipedia.org/wiki/Glycogenolysis

https://en.wikipedia.org/wiki/Fatty_acid_metabolism#Fatty_acid_catabolism

https://en.wikipedia.org/wiki/Liver

https://en.wikipedia.org/wiki/Renal_cortex

https://en.wikipedia.org/wiki/Kidney

https://en.wikipedia.org/wiki/Ruminants

https://en.wikipedia.org/wiki/Fasting

https://en.wikipedia.org/wiki/Starvation

https://en.wikipedia.org/wiki/Low-carbohydrate_diet

https://en.wikipedia.org/wiki/Exercise

https://en.wikipedia.org/wiki/Endergonic

https://en.wikipedia.org/wiki/Adenosine_triphosphate

https://en.wikipedia.org/wiki/Guanosine_triphosphate

https://en.wikipedia.org/wiki/Exergonic

https://en.wikipedia.org/wiki/Pyruvate

https://en.wikipedia.org/wiki/Glucose-6-phosphate

https://en.wikipedia.org/wiki/Ketosis




In ruminants, because dietary carbohydrates tend to be metabolized

by rumen organisms, gluconeogenesis occurs regardless of fasting, low-

carbohydrate diets, exercise, etc.

Pathway

Gluconeogenesis is a pathway consisting of a series of eleven enzyme-catalyzed

reactions. The pathway will begin in either the liver or kidney, in the

mitochondria or cytoplasm of those cells, this being dependent on the substrate

being used. Many of the reactions are the reverse of steps found in glycolysis.

https://en.wikipedia.org/wiki/Ruminant

https://en.wikipedia.org/wiki/Rumen




Gluconeogenesis begins in the mitochondria with the formation of oxaloacetate

by the carboxylation of pyruvate. This reaction also requires one molecule

of ATP, and is catalyzed by pyruvate carboxylase. This enzyme is stimulated by

high levels of acetyl-CoA (produced in β-oxidation in the liver) and inhibited by

high levels of ADP and glucose.

Oxaloacetate is reduced to malate using NADH, a step required for its

transportation out of the mitochondria.

Malate is oxidized to oxaloacetate using NAD+ in the cytosol, where the

remaining steps of gluconeogenesis take place.

Oxaloacetate is decarboxylated and then phosphorylated to

form phosphoenolpyruvate using the enzyme PEPCK. A molecule

of GTP is hydrolyzed to GDP during this reaction.

The next steps in the reaction are the same as reversed glycolysis.

However, fructose 1,6-bisphosphatase converts fructose 1,6-

bisphosphate to fructose 6-phosphate, using one water molecule and

releasing one phosphate (in glycolysis, phosphofructokinase 1 converts

F6P and ATP to F1,6BP and ADP). This is also the rate-limiting step of

gluconeogenesis.

Glucose-6-phosphate is formed from fructose 6-

phosphate by phosphoglucoisomerase (the reverse of step 2 in glycolysis).

Glucose-6-phosphate can be used in other metabolic pathways or

dephosphorylated to free glucose. Whereas free glucose can easily diffuse

in and out of the cell, the phosphorylated form (glucose-6-phosphate) is

locked in the cell, a mechanism by which intracellular glucose levels are

controlled by cells.

The final gluconeogenesis, the formation of glucose, occurs in

the lumen of the endoplasmic reticulum, where glucose-6-phosphate is

https://en.wikipedia.org/wiki/Pyruvate_carboxylase

https://en.wikipedia.org/wiki/Acetyl-CoA

https://en.wikipedia.org/wiki/Phosphoenolpyruvate

https://en.wikipedia.org/wiki/Glycolysis

https://en.wikipedia.org/wiki/Fructose_1,6-bisphosphate

https://en.wikipedia.org/wiki/Fructose_1,6-bisphosphate

https://en.wikipedia.org/wiki/Phosphofructokinase_1

https://en.wikipedia.org/wiki/Adenosine_triphosphate

https://en.wikipedia.org/wiki/Adenosine_diphosphate




hydrolyzed by glucose-6-phosphatase to produce glucose and release an

inorganic phosphate. Like two steps prior, this step is not a simple reversal

of glycolysis, in which hexokinase catalyzes the conversion of glucose and

ATP into G6P and ADP. Glucose is shuttled into the cytoplasm by glucose

transporters located in the endoplasmic reticulum's membrane.

Significance

The Enzyme Pyruvate carboxylase, a deficiency is seen as an inborn error

of metabolism, where mental retardation is manifested.

Its incidence is one in 25,000 births.

Pyruvate carboxylase gene is located in human chromosome No. 11.

In type II diabetes mellitus condition, the risen Gluconeogenesis is

responsible for the production of excessive Glucose after an overnight

fast. A continual supply of Glucose is necessary as a source of energy,

especially for the Nervous system and the Erythrocytes.

Gluconeogenesis mechanism is used to clear the products of the

metabolism of other tissues from the blood, eg: Lactate, produced by

Muscle and erythrocytes and Glycerol, which is continuously produced

by adipose tissue.




HORMONAL REGULATION OF BLOOD GLUCOSE LEVEL AND

DIABETES MELLITUS

Blood glucose regulation involves maintaining blood glucose levels at constant

levels in the face of dynamic glucose intake and energy use by the body.

Glucose is key in the energy intake of humans. On average this target range is

60-100 mg/dL for an adult although people can be asymptomatic at much more

varied levels. In order to maintain this range there are two main hormones that

control blood glucose levels: insulin and glucagon. Insulin is released when

there are high amounts of glucose in the blood stream.

Glucagon is released when there are low levels of glucose in the blood stream.

There are other hormones that effect glucose regulation and are mainly

controlled by the sympathetic nervous system. Blood glucose regulation is very

important to the maintenance of the human body. The brain doesn’t have any

energy storage of its own and as a result needs a constant flow of glucose, using

about 120 grams of glucose daily or about 60% of total glucose used by the

body at resting state. Without proper blood glucose regulation the brain and

other organs could starve leading to death.

Hormonal regulation of blood glucose level

Cells of the body require nutrients in order to function, and these nutrients are

obtained through feeding. In order to manage nutrient intake, storing excess

intake and utilizing reserves when necessary, the body uses hormones to

moderate energy stores. Insulin is produced by the beta cells of the pancreas,

which are stimulated to release insulin as blood glucose levels rise (for example,

after a meal is consumed). Insulin lowers blood glucose levels by enhancing the

rate of glucose uptake and utilization by target cells, which use glucose for ATP




production. It also stimulates the liver to convert glucose to glycogen, which is

then stored by cells for later use. Insulin also increases glucose transport into

certain cells, such as muscle cells and the liver. This results from an insulin-

mediated increase in the number of glucose transporter proteins in cell

membranes, which remove glucose from circulation by facilitated diffusion. As

insulin binds to its target cell via insulin receptors and signal transduction, it

triggers the cell to incorporate glucose transport proteins into its membrane.

This allows glucose to enter the cell, where it can be used as an energy source.

However, this does not occur in all cells: some cells, including those in the

kidneys and brain, can access glucose without the use of insulin. Insulin also

stimulates the conversion of glucose to fat in adipocytes and the synthesis of

proteins. These actions mediated by insulin cause blood glucose concentrations

to fall, called a hypoglycemic “low sugar” effect, which inhibits further insulin

release from beta cells through a negative f Impaired insulin function can lead

to a condition called diabetes mellitus, the main symptoms of which are

illustrated in Figure 1. This can be caused by low levels of insulin production by

the beta cells of the pancreas, or by reduced sensitivity of tissue cells to insulin.

This prevents glucose from being absorbed by cells, causing high levels of

blood glucose, or hyperglycemia (high sugar). High blood glucose levels make

it difficult for the kidneys to recover all the glucose from nascent urine,

resulting in glucose being lost in urine. High glucose levels also result in less

water being reabsorbed by the kidneys, causing high amounts of urine to be

produced; this may result in dehydration. Over time, high blood glucose levels

can cause nerve damage to the eyes and peripheral body tissues, as well as

damage to the kidneys and cardiovascular system. Over secretion of insulin can

cause hypoglycemia, low blood glucose levels. This causes insufficient glucose




availability to cells, often leading to muscle weakness, and can sometimes cause

unconsciousness or death if left untreated.

When blood glucose levels decline below normal levels, for example between

meals or when glucose is utilized rapidly during exercise, the

hormone glucagon is released from the alpha cells of the pancreas. Glucagon

raises blood glucose levels, eliciting what is called a hyperglycemic effect, by

stimulating the breakdown of glycogen to glucose in skeletal muscle cells and

liver cells in a process called glycogenolysis. Glucose can then be utilized as

energy by muscle cells and released into circulation by the liver cells. Glucagon

also stimulates absorption of amino acids from the blood by the liver, which

then converts them to glucose. This process of glucose synthesis is

called gluconeogenesis. Glucagon also stimulates adipose cells to release fatty

acids into the blood. These actions mediated by glucagon result in an increase in

blood glucose levels to normal homeostatic levels. Rising blood glucose levels

inhibit further glucagon release by the pancreas via a negative feedback

mechanism. In this way, insulin and glucagon work together to maintain

homeostatic glucose levels, as shown in Figure 2.




Regulation of Blood Glucose Levels by Thyroid Hormones

The basal metabolic rate, which is the amount of calories required by the body

at rest, is determined by two hormones produced by the thyroid

gland: thyroxine, also known as tetraiodothyronine or T4,

and triiodothyronine, also known as T3. These hormones affect nearly every

cell in the body except for the adult brain, uterus, testes, blood cells, and spleen.

They are transported across the plasma membrane of target cells and bind to

receptors on the mitochondria resulting in increased ATP production. In the

nucleus, T3 and T4activate genes involved in energy production and glucose

oxidation. This results in increased rates of metabolism and body heat

production, which is known as the hormone’s calorigenic effect.

T3 and T4 release from the thyroid gland is stimulated by thyroid-stimulating

hormone (TSH), which is produced by the anterior pituitary. TSH binding at

the receptors of the follicle of the thyroid triggers the production of T3 and

T4 from a glycoprotein called thyroglobulin. Thyroglobulin is present in the

follicles of the thyroid, and is converted into thyroid hormones with the addition

of iodine. Iodine is formed from iodide ions that are actively transported into the

thyroid follicle from the bloodstream. A peroxidase enzyme then attaches the

iodine to the tyrosine amino acid found in thyroglobulin. T3 has three iodine

ions attached, while T4 has four iodine ions attached. T3 and T4 are then released

into the bloodstream, with T4 being released in much greater amounts than T3.

As T3is more active than T4 and is responsible for most of the effects of thyroid

hormones, tissues of the body convert T4 to T3 by the removal of an iodine ion.

Most of the released T3 and T4 becomes attached to transport proteins in the

bloodstream and is unable to cross the plasma membrane of cells. These

protein-bound molecules are only released when blood levels of the unattached




hormone begin to decline. In this way, a week’s worth of reserve hormone is

maintained in the blood. Increased T3 and T4 levels in the blood inhibit the

release of TSH, which results in lower T3 and T4 release from the thyroid.

The follicular cells of the thyroid require iodides (anions of iodine) in order to

synthesize T3 and T4. Iodides obtained from the diet are actively transported into

follicle cells resulting in a concentration that is approximately 30 times higher

than in blood. The typical diet in North America provides more iodine than

required due to the addition of iodide to table salt. Inadequate iodine intake,

which occurs in many developing countries, results in an inability to synthesize

T3 and T4 hormones. The thyroid gland enlarges in a condition called goiter,

which is caused by overproduction of TSH without the formation of thyroid

hormone. Thyroglobulin is contained in a fluid called colloid, and TSH

stimulation results in higher levels of colloid accumulation in the thyroid. In the

absence of iodine, this is not converted to thyroid hormone, and colloid begins

to accumulate more and more in the thyroid gland, leading to goiter.

Disorders can arise from both the underproduction and overproduction of

thyroid hormones. Hypothyroidism, underproduction of the thyroid hormones,

can cause a low metabolic rate leading to weight gain, sensitivity to cold, and

reduced mental activity, among other symptoms. In children, hypothyroidism

can cause cretinism, which can lead to mental retardation and growth

defects. Hyperthyroidism, the overproduction of thyroid hormones, can lead to

an increased metabolic rate and its effects: weight loss, excess heat production,

sweating, and an increased heart rate. Graves’ disease is one example of a

hyperthyroid condition.




UNIT- II

Biological oxidation-reduction reactions

Oxidation-reduction (or "redox") reactions are a very large class of chemical

reactions in which both oxidation and reduction necessarily occur.

An oxidation is defined as loss of electrons in the course of a chemical reaction.

If a species gains electrons, it is undergoing a reduction. Since electrons are

"conserved" in a chemical reaction (they are not created or destroyed), one

chemical species' loss is another's gain. Thus, a reduction cannot occur with a

corresponding oxidation, and vice-versa. The term "redox" also nicely

encapsulates how inextricably tied together oxidation and reduction are in

reality.

Other terminology used in discussing redox chemistry: A chemical species that

gets reduced is acting as an oxidizing agent, or oxidant, while the species

undergoing oxidation is acting as the reducing agent, or reductant.

Oxidation state (or oxidation number) is a bookkeeping device employed by

chemists to help them classify and understand chemical reactions. The simplest

way to interpret oxidation number is to think of it as the number of electrons

lost or gained by an atom (compared to its neutral, uncombined form) when it

reacts to form ions or molecules. Consider first the case of ions. For monatomic

ions, such as Na+ or Cl−, the oxidation number is the same as the charge, +1 and

−1, for the sodium cation and chloride anion, respectively. In molecules and

polyatomic ions, oxidation states for atoms are calculated by comparing the

number of valence electrons in the neutral atom with a count of the surrounding

bonding and nonbonding electrons in the Lewis structure. In this respect, it is




similar to determining formal charge of an atom in a Lewis structure. A general

rule in determining an oxidation number of an atom in a complete Lewis

structure, is any differences in electronegativity between covalently bonded

atoms is treated as if the bond is actually ionic. That means both electrons are

counted as belonging to the more electronegative atom.

In computing formal charge, electrons in covalent bonds are treated as equally

shared, despite differences in electro negativity. But like formal charge, the sum

of the oxidation numbers for each atom in a formula or Lewis structure for a

molecular or ionic species must sum to the net charge of that formula or Lewis

structure (zero for a molecule). We will soon see that assignment of oxidation

numbers and following how they change in a chemical reaction allows us to

recognize redox reactions and determine the stoichiometry of the electron

transfer occurring.

It is easy to recognize any reaction featuring an uncombined, neutral element as

a redox reaction. Some examples are

CH4(g) + 2 O2(g) → CO2(g) + 2 H2O(l)

Fe(s) + O2(g) → Fe2O3(s)

2 Na(s) + Cl2(g) → 2 NaCl (s)

Identifying oxidation-reduction reactions using oxidation states

Applying these rules to the two previous reactions shows that the oxidation

states of O and H do not change from their usual values (−2, +1, respectively) in

either case. In the first reaction, tin is oxidized (its oxidation state is +2 on the

reactant side, and +4 on the product side, by rule 2), while the oxidation state of




N in the nitrate anion is +5, and in nitrogen monoxide it is +2 (by rules 3 and 4),

so nitrogen undergoes reduction. In the second reaction, chromium is reduced -

its oxidation state is +6 in the dichromate (Cr2O72−) anion (by rules 3 and 4) on

the reactant side, and is +3 (by rule 2) on the product side. The two carbon

atoms in ethanol can be assigned an oxidation state of −2 (it is actually an

average in this case - two atoms of the same element can have different

oxidation states depending on how they are bonded in the molecule or ion under

consideration, but that need not concern us further here), while in the product

acetic acid, we arrive at an oxidation state of zero for carbon. We see that the

organic compound ethanol is being oxidized to acetic acid by the oxidizing

agent dichromate.

Oxidation states of carbon atoms in simple organic compounds

The oxidation levels of carbon atoms in various functional groups will be

considered in order to train us to recognize oxidation and reduction in

biochemical reactions. The following shows how the oxidation number of the

carbon atom changes for the series of one-carbon molecules containing C, H,

and O only.

Note the oxidation number for carbon changes in steps of two in concert with

the addition or loss of two electrons and as the number of bonds the carbon

atom makes to oxygen decreases or increases.

Having assigned oxidation numbers, and understanding that changes in

oxidation numbers are the result of adding electrons (reduction) or removing

electrons (oxidation), balanced half reactions for oxidations or reductions can be

written. For example, the reduction of formaldehyde to methanol shown above

can first be written as an unbalanced reduction half reaction,




CH2O + 2e− → CH3OH

Since the equation is unbalanced with respect to charge and hydrogen atoms,

acidic conditions are typically assumed in cases such as this (meaning that we

add H+ on either side of the equation as required to balance both hydrogen

atoms and charge), and the balanced reduction half reaction can be written:

CH2O + 2H+ + 2e− → CH3OH

Reactive oxygen species

A similar series for the simplest oxygen compounds is of biological importance.

The energy-yielding process of oxidative phosphorylation results in the

reduction of molecular oxygen to water. Both oxygen and water are themselves

benign, such is not true for intermediates that are only partially reduced to

water. Although the molecular choreography of oxygen reduction in cells that

carry out oxidative metabolism is tightly and precisely channeled so as to

minimize the possibility of their release, occasionally and inevitably a partially

reduced oxygen species is produced.

Redox chemistry and electricity

Charge (q) is measured in Coulombs (C)

Charge of an electron: 1.60217653 × 10−19 C

The Faraday (F), the charge of 1 mol elementary charge:

F = 9.648534 × 104 C · mol−1 = 96.485 kJ ·V−1· mol−1




Current (I) is measured in amperes (A). An ampere is defined as 1 A = 1 C ·

s −1

Voltage and electrical work. The flow of water is a useful analogy for electric

current. The flow as measured by volume of water per unit time would be like

the flow of charge per unit time. Furthermore, the gravitational potential

difference (in height of a column or reservoir of water above the level where

flow is measured) is analogous to the electric potential difference existing

between two charge "reservoirs". When charge flows "downhill" through an

electric potential gradient, it can do useful work, just as a mass of water flowing

downhill toward lower gravitational potential can do the work of turning a

turbine (and thereby generating ....electric potential energy!). Electric potential

is measured in volts, where a volt is defined as 1 V = 1 J · C −1 . In other words,

a charge of 1 C moving through a potential difference of 1 V is equivalent to 1 J

of work (work is equivalent to energy, as shown by the work-energy theorem of

physics). Note that 1 J · V −1 = 1 C is a convenient conversion factor for charge.

(This is used above in the unit conversion for the Faraday constant.) For reasons

of clarity, the potential difference quantity, also referred to as electromotive

force (emf), will be represented here as ΔE.

Redox processes and electron carrier molecules in biochemistry

A great many biochemical reactions are oxidation-reduction reactions, so in a

sense the participants in these reactions that undergo oxidation or reduction are

electron carriers. In order for redox processes to serve as a source of energy in

the form of an emf through which electrons flow, organisms utilize

(heterotrophs) or are able to create (autotrophs) molecules that are reducing

agents. These electrons get passed from one electron carrier to the next, in a

series of electron transfer reactions. Electrons flow exergonically from species




with a lower to a higher reduction potential. Reduction of a terminal electron

acceptor, such as O2 in aerobic metabolism yields an end product which is

exchanged with surroundings.

There are recurring patterns of redox reactions in biochemistry. Metabolic

intermediates that are relatively reduced, derived for example from the the food

consumed by heterotrophic organisms, such as glucose 6-phosphate or pyruvate,

can be oxidized with electrons being transferred to the oxidized forms of redox

cofactors that serve as modular cosubstrates in enzyme-catalyzed redox

reactions. The enzymes involved are in the oxidoreductase class of enzymes,

and many of these work with one of two modular cosubstrates, nicotinamide

adenine dinucleotide and flavin adenine dinucleotide.

Nicotinamide adenine dinucleotide (NAD) is an important, ubiquitous redox

cofactor that functions as a carrier of electron pairs. The oxidized form of the

cofactor carries a positive charge, and is denoted NAD+ while the reduced form

is NADH. The nicotinamide portion of NAD+, consisting of a carbamylated

pyridine ring (in red in the figure below, corresponding to niacin, one of the B-

complex vitamins acts as the electron pair acceptor. In undergoing reduction,

the 4-position of the NAD+ ring (para to the nitrogen atom) in effect accepts a

hydride ion (H:−). Note that the "dinucleotide" part of the name is due to the fact

that the other "half" of NAD is an adenine-containing nucleotide, AMP. The

two "halves" of the molecule are linked by a phosphoanhydride bond.




A closely related cofactor, NADP+, (reduced form, NADPH) differs only in

having a phosphate attached to the 2′ position of the ribose attached to adenine.

NADPH generally functions in redox reactions in biosynthetic pathways (e.g.

fatty acid synthesis, whereas NAD+ predominates in catabolic processes, such

as those associated with glycolysis and oxidative phosphorylation.

The NAD+/NADH redox half-reaction (see figure below) has a standard

biochemical reduction potential, ΔE°′ of −0.315 V. The progress of reactions

involving NAD+/NADH can be conveniently monitored spectrophotometrically

due to the appearance of a broad absorption with its peak at 340 nm

when NADH is formed.

The major source of NADH in oxidative metabolism is the citric acid

cycle. The NADH produced is reoxidized to NAD+ when the former donates its

http://guweb2.gonzaga.edu/faculty/cronk/CHEM245pub/phosphoryl.html

http://guweb2.gonzaga.edu/faculty/cronk/CHEM245pub/glycolysis.html




electrons to the first component of the electron transport chain (ETC).

Eventually these electrons reduce molecular oxygen to water.

Flavin adenine dinucleotide (FAD) is an important, ubiquitous redox cofactor,

consisting of (like NAD) an AMP moiety in an anhydride linkage

to FMN (flavin mononucleotide, shown in blue in the figure).

The isoalloxazine ring system of FAD or FMN can accept one or two electrons,

in contrast to NAD, which can only be reduced by two electrons at a time. In

the electron transport chain (ETC) FMN acts as a cofactor in the NADH-Q

oxidoreductase complex, accepting 2 electrons from NADH, and transferring

them to a series of iron-sulfur (Fe-S) proteins, and then to coenzyme Q

(ubiquinone). Electrons from FADH2 enter the electron transport chain at the

level of cytochrome reductase, the second proton-pumping complex in the

electron transport chain (downstream from NADH-Q reductase), via

the succinate-Q oxidoreductase complex, which accomplishes the transfer of

electrons from FADH2 to coenzyme Q (again through Fe-S proteins). The full

structure of FAD is shown below. The three rings at the top constitute the

isoalloxazine ring system, or flavin portion of the molecule. The portion of the

molecule corresponding to FMN (shown in blue) also includes the residue of the

five-carbon D-ribitol (a polyhydroxy alcohol derived from the sugar D-

ribose with an attached phosphate group. The isoalloxazine ring plus ribitol

corresponds to riboflavin, one of the B-complex vitamins.




FMN is linked to an adenosine monophosphate (AMP) shown in black) by a

phosphodiester bond. The reduction of FAD involves the 1 and 5

nitrogen atoms (labelled red in the figure at left), and the oxidation states of

FAD/FMN are shown in the figure below. The molecule labelled (1) represents

FAD or FMN - the most oxidized form. The molecule labelled (2) is a radical

or semiquinone formed by a one electron reduction of (1). A second one-

electron reduction converts the radical to (3), which represents the fully reduced

forms FADH2 or FMH2.




Electron Transport Chain

Definition

The electron transport chain is a cluster of proteins that transfer electrons

through a membrane within mitochondria to form a gradient of protons that

drives the creation of adenosine triphosphate (ATP). ATP is used by the cell as

the energy for metabolic processes for cellular functions.

During the process, a proton gradient is created when the protons are pumped

from the mitochondrial matrix into the intermembrane space of the cell, which

also helps in driving ATP production. Often, the use of a proton gradient is

referred to as the chemiosmotic mechanism that drives ATP synthesis since it

relies on a higher concentration of protons to generate “proton motive force”.

The amount of ATP created is directly proportional to the number of protons

that are pumped across the inner mitochondrial membrane.

The electron transport chain involves a series of redox reactions that relies on

protein complexes to transfer electrons from a donor molecule to an acceptor

molecule. As a result of these reactions, the proton gradient is produced,

enabling mechanical work to be converted into chemical energy, allowing ATP

synthesis. The complexes are embedded in the inner mitochondrial membrane

called the cristae in eukaryotes. Enclosed by the inner mitochondrial membrane

is the matrix, which is where necessary enzymes such

as pyruvate dehydrogenase and pyruvate carboxylase are located. The process

can also be found in photosynthetic eukaryotes in the thylakoid membrane of

chloroplasts and in prokaryotes, but with modifications.

https://biologydictionary.net/cristae/

https://biologydictionary.net/pyruvate/




By-products from other cycles and processes, like the citric acid cycle, amino

acid oxidation, and fatty acid oxidation, are used in the electron transport chain.

As seen in the overall redox reaction,

2 H+ + 2 E+ + ½ O2 → H2O + ENERGY

energy is released in an exothermic reaction when electrons are passed through

the complexes; three molecules of ATP are created. Phosphate located in the

matrix is imported via the proton gradient, which is used to create more ATP.

The process of generating more ATP via the phosphorylation of ADP is referred

to oxidative phosphorylation since the energy of hydrogen oxygenation is used

throughout the electron transport chain. The ATP generated from this reaction

go on to power most cellular reactions necessary for life.

Mechanism of ETC

In the electron transfer chain, electrons move along a series of proteins to

generate an expulsion type force to move hydrogen ions, or protons, across the

mitochondrial membrane. The electrons begin their reactions in Complex I,

continuing onto Complex II, traversed to Complex III and cytochrome c

via coenzyme Q, and then finally to Complex IV. The complexes themselves

are complex-structured proteins embedded in the phospholipid membrane. They

are combined with a metal ion, such as iron, to help with proton expulsion into

the intermembrane space as well as other functions. The complexes also

undergo conformational changes to allow openings for the transmembrane

movement of protons.

These four complexes actively transfer electrons from an organic metabolite,

such as glucose. When the metabolite breaks down, two electrons and a

https://biologydictionary.net/oxidative-phosphorylation/

https://biologydictionary.net/phospholipid/




hydrogen ion are released and then picked up by the coenzyme NAD+ to

become NADH, releasing a hydrogen ion into the cytosol.

Fig.1 Electron transport chain

The NADH now has two electrons passing them onto a more mobile molecule,

ubiquinone (Q), in the first protein complex (Complex I). Complex I, also

known as NADH dehydrogenase, pumps four hydrogen ions from the matrix

into the intermembrane space, establishing the proton gradient. In the next

protein, Complex II or succinate dehydrogenase, another electron carrier and

coenzyme, succinate is oxidized into fumarate, causing FAD (flavin-adenine

dinucleotide) to be reduced to FADH2. The transport molecule, FADH2 is then

reoxidized, donating electrons to Q (becoming QH2), while releasing another

hydrogen ion into the cytosol. While Complex II does not directly contribute to

the proton gradient, it serves as another source for electrons.

https://biologydictionary.net/cytosol/




Fig.2 Electron transport chain in cell membrane

Complex III, or cytochrome c reductase, is where the Q cycle takes place. There

is an interaction between Q and cytochromes, which are molecules composed of

iron, to continue the transfer of electrons. During the Q cycle, the ubiquinol

(QH2) previously produced donates electrons to ISP and cytochrome b

becoming ubiquinone. ISP and cytochrome b are proteins that are located in the

matrix that then transfers the electron it received from ubiquinol to cytochrome

c1. Cytochrome c1 then transfers it to cytochrome c, which moves the electrons

to the last complex. (Note: Unlike ubiquinone (Q), cytochrome c can only carry

one electron at a time). Ubiquinone then gets reduced again to QH2, restarting




the cycle. In the process, another hydrogen ion is released into the cytosol to

further create the proton gradient.

The cytochromes then extend into Complex IV, or cytochrome c oxidase.

Electrons are transferred one at a time into the complex from cytochrome c. The

electrons, in addition to hydrogen and oxygen, then react to form water in an

irreversible reaction. This is the last complex that translocates four protons

across the membrane to create the proton gradient that develops ATP at the end.

As the proton gradient is established, F1F0 ATP synthase, sometimes referred to

as Complex V, generates the ATP. The complex is composed of several

subunits that bind to the protons released in prior reactions. As the protein

rotates, protons are brought back into the mitochondrial matrix, allowing ADP

to bind to free phosphate to produce ATP. For every full turn of the protein,

three ATP is produced, concluding the electron transport chain.

Oxidative phosphorylation

Definition

Oxidative phosphorylation (UK or electron transport-linked phosphorylation) is

the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby

releasing the chemical energy of molecular oxygen, which is used to produce

adenosine triphosphate (ATP). In most eukaryotes, this takes place inside

mitochondria. Almost all aerobic organisms carry out oxidative

phosphorylation. This pathway is so pervasive because the energy of the double

bond of oxygen is so much higher than the energy of the double bond in

carbondioxide or in pairs of single bonds in organic molecules observed in

alternative fermentation processes such as anaerobic glycolysis.




Overview:

Fig. 3 Simple diagram of the electron transport chain

The electron transport chain is a series of proteins embedded in the inner

mitochondrial membrane.

In the matrix, NADH and FADH2 deposit their electrons in the chain (at the

first and second complexes of the chain, respectively).

The energetically "downhill" movement of electrons through the chain causes

pumping of protons into the intermembrane space by the first, third, and fourth

complexes.

Finally, the electrons are passed to oxygen, which accepts them along with

protons to form water.

The proton gradient produced by proton pumping during the electron transport

chain is used to synthesize ATP. Protons flow down their concentration gradient




into the matrix through the membrane protein ATP synthase, causing it to spin

(like a water wheel) and catalyze conversion of ADP to ATP.

The electron transport chain is a series of proteins and organic molecules

found in the inner membrane of the mitochondria. Electrons are passed from

one member of the transport chain to another in a series of redox reactions.

Energy released in these reactions is captured as a proton gradient, which is then

used to make ATP in a process called chemiosmosis. Together, the electron

transport chain and chemiosmosis make up oxidative phosphorylation. The

key steps of this process, shown in simplified form in the diagram above,

include:

Delivery of electrons by NADH and FADH_22start subscript, 2, end

subscript. Reduced electron carriers (NADH and FADH_22start

subscript, 2, end subscript) from other steps of cellular respiration transfer

their electrons to molecules near the beginning of the transport chain. In

the process, they turn back into NAD^++start superscript, plus, end

superscript and FAD, which can be reused in other steps of cellular

respiration.

Electron transfer and proton pumping. As electrons are passed down

the chain, they move from a higher to a lower energy level, releasing

energy. Some of the energy is used to pump H^++start superscript, plus,

end superscript ions, moving them out of the matrix and into the

intermembrane space. This pumping establishes an electrochemical

gradient.

Splitting of oxygen to form water. At the end of the electron transport

chain, electrons are transferred to molecular oxygen, which splits in half

and takes up H^++start superscript, plus, end superscript to form water.




Gradient-driven synthesis of ATP. As H^++start superscript, plus, end

superscript ions flow down their gradient and back into the matrix, they

pass through an enzyme called ATP synthase, which harnesses the flow

of protons to synthesize ATP.

We'll look more closely at both the electron transport chain and chemiosmosis

in the sections below.

Substrate-level phosphorylation

Substrate-level phosphorylation is a metabolic reaction that results in the

formation of ATP or GTP by conversion of a higher energy substrate (whether

phosphate group attached or not) into lower energy product and a using some of

the released chemical energy, the Gibbs free energy, to transfer

a phosphoryl (PO3) group to ADP or GDP from another phosphorylated

compound.

Unlike oxidative phosphorylation, oxidation and phosphorylation are not

coupled in the process of substrate-level phosphorylation, and reactive

intermediates are most often gained in the course of oxidation processes

in catabolism. Most ATP is generated by oxidative phosphorylation in aerobic

or anaerobic respiration while substrate-level phosphorylation provides a

quicker, less efficient source of ATP, independent of external electron

acceptors. This is the case in human erythrocytes, which have no mitochondria,

and in oxygen-depleted muscle.




Adenosine triphosphate is a major "energy currency" of the cell. The high

energy bonds between the phosphate groups can be broken the power a variety

of reactions used in all aspects of cell function.

Substrate-level phosphorylation occurs in the cytoplasm of cells

during glycolysis and in mitochondria either during the Krebs cycle or

by MTHFD1L , an enzyme interconverting ADP + phosphate + 10-

formyltetrahydrofolate to ATP + formate + tetrahydrofolate (reversibly), under

both aerobic and anaerobic conditions. In the pay-off phase of glycolysis, a net

of 2 ATP are produced by substrate-level phosphorylation.

Glycolysis

The first substrate-level phosphorylation occurs after the conversion of 3-

phosphoglyceraldehyde and Pi and NAD+ to 1,3-bisphosphoglycerate

via glyceraldehyde 3-phosphate dehydrogenase. 1,3-bisphosphoglycerate is then

dephosphorylated via phosphoglycerate kinase, producing 3-phosphoglycerate

and ATP through a substrate-level phosphorylation.

The second substrate-level phosphorylation occurs by

dephosphorylating phosphoenolpyruvate, catalyzed by pyruvate kinase,

producing pyruvate and ATP.

During the preparatory phase, each 6-carbon glucose molecule is broken into

two 3-carbon molecules. Thus, in glycolysis dephosphorylation results in the

production of 4 ATP. However, the prior preparatory phase consumes 2 ATP,

so the net yield in glycolysis is 2 ATP. 2 molecules of NADH are also produced

and can be used in oxidative phosphorylation to generate more ATP.

https://en.wikipedia.org/wiki/Glyceraldehyde_3-phosphate_dehydrogenase

https://en.wikipedia.org/wiki/Phosphoglycerate_kinase

https://en.wikipedia.org/w/index.php?title=Phosphenolpyruvate&action=edit&redlink=1

https://en.wikipedia.org/wiki/Pyruvate_kinase

https://en.wikipedia.org/wiki/Pyruvate




Mitochondria

ATP can be generated by substrate-level phosphorylation in mitochondria in a

pathway that is independent from the proton motive force. In the matrix there

are three reactions capable of substrate-level phosphorylation, utilizing

either phosphoenolpyruvate carboxykinase or succinate-CoA ligase,

or monofunctional C1-tetrahydrofolate synthase.

Phosphoenolpyruvate carboxykinase

Mitochondrial phosphoenolpyruvate carboxykinase is thought to participate in

the transfer of the phosphorylation potential from the matrix to the cytosol and

vice versa. However, it is strongly favored towards GTP hydrolysis, thus it is

not really considered as an important source of intra-mitochondrial substrate-

level phosphorylation.

Succinate-CoA ligase

Succinate-CoA ligase is a heterodimer composed of an invariant α-subunit and a

substrate-specific ß-subunit, encoded by either SUCLA2 or SUCLG2. This

combination results in either an ADP-forming succinate-CoA ligase or a GDP-

forming succinate-CoA ligase. The ADP-forming succinate-CoA ligase is

potentially the only matrix enzyme generating ATP in the absence of a proton

motive force, capable of maintaining matrix ATP levels under energy-limited

conditions, such as transient hypoxia.

Monofunctional C1-tetrahydrofolate synthase

This enzyme is encoded by MTHFD1L and reversibly interconverts ADP +

phosphate + 10-formyltetrahydrofolate to ATP + formate + tetrahydrofolate.

https://en.wikipedia.org/wiki/Phosphoenolpyruvate_carboxykinase

https://en.wikipedia.org/wiki/Succinyl_coenzyme_A_synthetase




Other mechanisms

In working skeletal muscles and the brain, Phosphocreatine is stored as a readily

available high-energy phosphate supply, and the enzyme creatine

phosphokinase transfers a phosphate from phosphocreatine to ADP to produce

ATP. Then the ATP releases giving chemical energy. This is sometimes

erroneously considered to be substrate-level phosphorylation, although it is

a transphosphorylation.

Importance of substrate-level phosphorylation in anoxia

During anoxia, provision of ATP by substrate-level phosphorylation in the

matrix is important not only as a mere means of energy, but also to prevent

mitochondria from straining glycolytic ATP reserves by maintaining

the adenine nucleotide translocator in ‘forward mode’ carrying ATP towards the

cytosol.

Oxidative phosphorylation

An alternative method used to create ATP is through oxidative phosphorylation,

which takes place during cellular respiration. This process utilizes the oxidation

of NADH to NAD+, yielding 3 ATP, and of FADH2 to FAD, yielding 2 ATP.

The potential energy stored as an electrochemical gradient of protons (H+)

across the inner mitochondrial membrane is required to generate ATP from

ADP and Pi (inorganic phosphate molecule), a key difference from substrate-

level phosphorylation. This gradient is exploited by ATP synthase acting as a

pore, allowing H+ from the mitochondrial intermembrane space to move down

its electrochemical gradient into the matrix and coupling the release of free

https://en.wikipedia.org/wiki/Phosphocreatine

https://en.wikipedia.org/wiki/Creatine_phosphokinase

https://en.wikipedia.org/wiki/Creatine_phosphokinase

https://en.wikipedia.org/wiki/Transphosphorylation

https://en.wikipedia.org/wiki/Hypoxia_(medical)

https://en.wikipedia.org/wiki/Adenine_nucleotide_translocator

https://en.wikipedia.org/wiki/Oxidative_phosphorylation

https://en.wikipedia.org/wiki/Cellular_respiration

https://en.wikipedia.org/wiki/NADH

https://en.wikipedia.org/wiki/Potential_energy

https://en.wikipedia.org/wiki/Electrochemical_gradient

https://en.wikipedia.org/wiki/ATP_synthase

https://en.wikipedia.org/wiki/Intermembrane_space




energy to ATP synthesis. Conversely, electron transfer provides the energy

required to actively pump H+ out of the matrix.

Uncouplers of oxidative phosphorylation

Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling

between the electron transport and phosphorylation reactions and thus inhibit

ATP synthesis without affecting the respiratory chain and ATP synthase.

Uncouplers inhibit ATP synthesis by preventing this coupling reaction in such a

fashion that the energy produced by redox reactions cannot be used for

phosphorylation. Uncouplers include DNP, valinomycin, and CCCP. Most of

them are hydrophobic weak acids that act by protonophoric action and

activities.

Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling

between the electron transport and phosphorylation reactions and thus inhibit

ATP synthesis without affecting the respiratory chain and ATP synthase (H(+)-

ATPase). Miscellaneous compounds are known to be uncouplers, but weakly

acidic uncouplers are representative because they show very potent activities.

The most potent uncouplers discovered so far are the hindered phenol SF 6847,

and hydrophobic salicylanilide S-13, which are active in vitro at concentrations

in the 10 nM range. For induction of uncoupling, an acid dissociable group,

bulky hydrophobic moiety and strong electron-withdrawing group are required.

Weakly acidic uncouplers are considered to produce uncoupling by their

protonophoric action in the H(+)-impermeable mitochondrial membrane. For

exerting these effects, the stability of the respective uncoupler anions in the

hydrophobic membrane is very important. High stability is achieved by

delocalization of the polar ionic charge through uncoupler (chemical)-specific

mechanisms. Such an action of weakly acidic uncouplers is characteristic of the




highly efficient membrane targeting action of a nonsite-specific type of

bioactive compound.

One example of an ‘uncoupler’ of oxidative phosphorylation is DNP (2,4-

dinitrophenol).

2,4-Dinitrophenol (DNP), C6H4N2O5, is a cellular metabolic poison. It

uncouples oxidative phosphorylation by carrying protons across the

mitochondrial membrane, leading to a rapid consumption of energy without

generation of ATP.

In living cells, DNP acts as a proton ionophore, an agent that can shuttle protons

(hydrogen ions) across biological membranes. It defeats the proton gradient

across mitochondrial membrane, collapsing the proton motive force that the cell

uses to produce most of its ATP chemical energy. Instead of producing ATP,

the energy of the proton gradient is lost as heat.

DNP is often used in biochemistry research to help explore the bioenergetics of

chemiosmotic and other membrane transport processes.

The HMP shunt represents an alternative pathway for the breakdown of glucose.

Briefly describe the main products produced by this pathway and it’s biological

significance.

The main product are Ribose-5-P, NADPH and Intermediates of the glycolytic

pathway. HMP shunt represents an alternate degradative pathway for the

breakdown of glucose, and it provides a link between glycolysis and nucleotide

metabolism and fatty acid.




Biological significance of Ribose-5-P is that serves as the precursor to various

nucleotides (ATP, NAD, NADP, coenzyme A) and nucleic acids (DNA) within

our cells.

Biological significance of NADPH: represents the major source of reducing

power for biosynthetic reactions within cells, particularly the synthesis of fatty

acids. It follows that the HMP shunt is active in tissues specialized for the

synthesis of fatty acids or steroids.

Biological significance of Intermediates of the glycolytic pathway: the demand

for NADPH in the cell is usually far greater than the demand for ribose-5-P,

thus the second phase of this pathway is devoted to recycling the 5-carbon

skeletons into intermediates of the glycolytic pathway so that the cell can

harness the energy that is present in these molecules.

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