GLYCOLYSIS

Glycolysis comes from a merger of two Greek words: Glykys = sweet and Lysis = breakdown/ splitting
It is also known as Embden-Meyerhof-Parnas pathway or EMP pathway.
Glycolysis is the sequence of 10 enzyme-catalyzed reactions that converts glucose into pyruvate with simultaneous
production of ATP. In this oxidative process, 1mol of glucose is partially oxidised to 2 moles of pyruvate. This major
pathway of glucose metabolism occurs in the cytosol of nearly all cells. It occurs aerobically as well as anaerobically as it
doesn’t directly involve molecular oxygen.

The glycolytic breakdown of glucose is the sole source of metabolic energy in some mammalian tissues and cell types
(erythrocytes, renal medulla, brain, and sperm, for example). Some plant tissues that are modified to store starch
(such as potato tubers) and some aquatic plants (e.g watercress) derive most of their energy from glycolysis; many
anaerobic microorganisms are entirely dependent on glycolysis.

The glycolytic sequence of reactions differs from species to species only in the mechanism of its regulation and in the
subsequent metabolic fate of the pyruvate formed. In aerobic organisms, glycolysis is the prelude to Citric acid cycle and
Electron Transport Chain. Glycolysis is the central pathway for Glucose catabolism.

During the sequential reactions of glycolysis, some of the free energy released from glucose is conserved in the form of
ATP and NADH.

Glycolysis leads to breakdown of 6-C glucose into two molecules of 3-C pyruvate with the enzyme catalyzed reactions
categorized into 2 phases:
1.Phase 1- preparatory phase
2.Phase 2- payoff phase.

PREPARATORY PHASE
It consists of the 1st 5 steps of glycolysis in which the glucose is enzymatically phosphorylated by ATP to yield Fructose-
1,6-biphosphate.
This fructuse-1,6-biphosphate is then split in half to yield 2 molecules of 3-carbon containing Glyceraldehyde-3-
phosphate/ dihyroxyacteone phosphate.
Thus the first phase results in cleavage of the hexose chain. This cleavage requires an investment of 2 ATP molecules to
activate the glucose mole and prepare it for its cleavage into 3-carbon compound
PAYOFF PHASE
This phase constitutes the last 5 reactions of Glycolysis.
This phase marks the release of ATP molecules during conversion of Glyceraldehyde-3-phosphtae to 2 moles of
Pyruvate.

Here 4 moles of ADP are phosphorylated to ATP. Although 4 moles of ATP are formed, the net result is only 2 moles of
ATP per mole of Glucose oxidized, since 2 moles of ATP are utilized in Phase 1

STEP 1: PHOSPHORYLATION
Glucose is phosphorylated by ATP to form sugar phosphate. This is an irreversible reaction & is catalyzed by hexokinase.
Thus the reaction can be represented as follows:
STEP 2: ISOMERIZATION:
It is a reversible rearrangement of chemical structure of carbonyl oxygen from C1 to C2, forming a Ketose from the
Aldose. Thus, isomerization of the aldose Glucose6-phosphate gives the ketose, Fructose-6-phoshphate

STEP 3: PHOPHORYLATION:
Here the Fructose-6-phosphate is phosphorylated by ATP to fructose-1,6-bisphosphate. This is an irreversible reaction
and is catalyzed by phosphofructokinase 1 (PFK-1) enzyme.

STEP 4: BREAKDOWN:
This six carbon sugar is cleaved to produce two 3-C molecules: glyceradldehyde-3-phosphate (GAP) & dihydroxyacetone
phosphate(DHAP). This reaction is catalyzed by Aldolase

STEP 5: ISOMERIZATION:
Dihydroxyacetone phosphate is oxidized to form Glyceraldehyde-3-phosphate. This reaction is catalyzed by triose
phosphate isomerase enzyme.

STEP 6: OXIDATION
2 molecules of Glyceraldehyde-3-phosphate are oxidized. Glyceraldehyde-3-phosphate dehydrogenase catalyzes the
conversion of Glyceraldehyde3-phosphate into 1,3-bisphosphoglycerate.

STEP 7: 1ST SUBSTRATE LEVEL PHOSPHORYLATION

The transfer of high-energy phosphate group that was generated earlier to ADP, form ATP. This phosphorylation i.e.
addition of phosphate to ADP to give ATP is termed as substrate level phosphorylation as the phosphate donor is the
substrate 1,3-bisphosphoglycerate (1,3-BPG). The product of this reaction is 2 molecules of 3-phosphoglycerate.

STEP 8: REARRANGEMENT
The remaining phosphate-ester linkage in 3-phosphoglycerate, is moved from carbon 3 to carbon 2, because of
relatively low free energy of hydrolysis, to form 2-phosphoglycerate(2-PG).

STEP 9: DEHYDRATION OF 2-PG

This is the second reaction in glycolysis where a high-energy phosphate compound is formed. The 2-phosphoglycerate is
dehydrated by the action of enolase to phosphoenolpyruvate(PEP). This compound is the phosphate ester of the enol
tautomer of pyruvate.This is a reversible reaction

STEP 10: 2ND SUBSTRATE LEVEL PHOSPHORYLATION (TRANSFER OF PHOSPHATE FROM PEP to ADP)
This last step is the irreversible transfer of high energy phosphoryl group from phosphoenolpuruvate to ADP.This
reaction is catalyzed by pyruvate kinase.This is the 2nd substrate level phosphorylation reaction in glycolysis which
yields ATP. This is a non-oxidative phosphorylation reaction.

Each molecule of glucose gives 2 molecules of Glyceraldehyde-3-phosphate which eventually becomes 2 molecules of
pyruvate. Therefore , the total input of all 10 reactions can be summarized as:

Glucose + 2 ATP+ 2Pi+ 2NAD⁺+ 2H⁺+ 4ADP 2 Pyruvate+ 2NADH+ 2ATP+ 2H⁺ + 2H₂O

Therefore in glycolysis, while Glucose is oxidized to Pyruvate, NAD⁺ is reduced to NADH and ADP is phosphorylated to
ATP at the same time in a stepwise series of reactions
Glycolysis releases only a small fraction of the total available energy of the glucose molecule; the two molecules of
pyruvate formed by glycolysis still contain most of the chemical potential energy of glucose, that energy can be
extracted by oxidative reactions in the citric acid cycle and oxidative phosphorylation.

Regulation of glycolysis:
The regulation of glycolysis is achieved by an interplay between ATP consumption, NADH regeneration, and
allosteric regulation of several glycolytic enzymes—including hexokinase, PFK-1, and pyruvate kinase—as well as
by fluctuations in the concentration of key metabolites that reflect the cellular balance between
ATP production and consumption. On a slightly longer time scale, glycolysis is regulated by the hormones
glucagon, epinephrine, and insulin, and by changes in the expression of the genes for several glycolytic
enzymes.

The irreversible reactions of the glycolytic pathway are the major regulatory points. The enzymes catalyzing these
reactions are hexokinase, PFK-1 and pyruvate kinase. These enzymes are subject to;
(i) allosteric control (the transient binding of molecules to the enzyme to change the conformation), effect is observed in
milliseconds.
(ii)covalent modification- which is generally phosphorylation and de-phosphorylation of the enzymes often mediated by
hormones. Effect is in seconds.
(iii)genetic control by of regulation transcription. Effect is observed in hours.

The 3 Points Of Control

1. Hexokinase and glucokinase catalyzes reaction # 1(conversion of glucose to glucose-6-phosphate).
Allows for the phosphorylation of glucose when blood levels of glucose are low. Hexokinase is strongly inhibited by its
product, glucose-6-Phosphate (G6P), while Glucokinase is inhibited by fructose-6-phosphate (F6P) [which is generated
by reaction # 2] and stimulated by fructose-1-phosphate (F1P).

2. Phosphofructo-1-kinase (PFK-1) catalyzes reaction # 3 catalyzes the conversion of fructose-6-phosphate to

fructose-1,6-bisphosphate. This is the rate-limiting step of glycolysis. First committed step of glycolysis. Subject to the
greatest degree of regulation by allosteric effectors. Positive effectors: low energy state of the cell signified by ADP, Pi
and AMP are positive effectors of PFK-1 as well as Fructose 2,6-bisphosphate(a product PFK-2)
Negative effectors. High energy state of the cell signified by ATP: ATP is an allosteric inhibitor of PFK-1 . Citrate, an
intermediate in citric acid cycle, is another allosteric inhibitor of PFK-1.

3. Pyruvate kinase catalyzes reaction # 10 which is the conversion of PEP to Pyruvate.

Inhibited by high concentrations of ATP, acetyl co A and fatty acids. Isoenzyme found in liver is activated by fructose 1,6-
bisphosphate. Liver enzyme is subject to phosphorylation.
Active in the dephosphorylated state. Inactive in the phosphorylated state. This enzyme is also regulated at the genetic
level
 The FATE of pyruvate

Lactate formation: Under anaerobic conditions or in the absence of mitochondria (as in erythrocytes, cornea and lens ),
pyruvate is reduced to lactate to regenerate NAD+.
When pyruvate cannot be oxidized within mitochondria for some reason (e.g. hypoxia, genetic defects in pyruvate
dehydrogenase or citric acid cycle enzymes, genetic defects in electron transport chain), pyruvate is reduced to lactate
by lactate dehydrogenase.

Anaerobic Ethanol Fermentation: Yeast and other microorganisms ferment glucose to ethanol and CO2, rather than to
lactate. Glucose is converted to pyruvate by glycolysis, and the pyruvate is converted to ethanol and CO2 in a two-step
process:
In the first step, pyruvate is decarboxylated in an irreversible reaction catalyzed by pyruvate decarboxylase.
This reaction is a simple decarboxylation and does not involve the net oxidation of pyruvate. Pyruvate decarboxylase
requires Mg2 and has a tightly bound coenzyme, thiamine pyrophosphate. In the second step, acetaldehyde is reduced
to ethanol through the action of alcohol dehydrogenase, with the reducing power furnished by NADH . Ethanol and CO2
are thus the end products of ethanol fermentation.
Under aerobic conditions, pyruvate can be oxidatively de-carboxylated to acetylCo-A and CO2 by a special enzyme
known as the pyruvate dehydrogenase complex (PDH), a cluster of enzymes—multiple copies of each
of three enzymes—located in the mitochondria of eukaryotic cells and in the cytosol of prokaryotes. The reaction is an
irreversible oxidation process in which the carboxyl group is removed from pyruvate as a molecule of CO2 and the two
remaining carbons become the acetyl group of acetyl-CoA.