Glycolysis & the Oxidation of Pyruvate

The reactions of glycolysis constitute the main pathway of glucose utilization.

All of the enzymes of glycolysis are cytosolic.
Glycolysis involves 10 enzymatic reactions:
1. The phosphorylation of glucose at position 6 forming glucose-6-phosphate
(G-6-P), by hexokinase.

Under physiological conditions, this reaction is irreversible. Hexokinase has a

high affinity (low Km) for glucose, and it is saturated under normal conditions.
Hexokinase is inhibited allosterically by its product, glucose-6-phosphate. In
tissues other than the liver (and pancreatic β -islet cells), the availability of glucose
for glycolysis is controlled by transport into the cell, which in turn is regulated by
insulin.
Hexokinase has a high affinity (low Km) for glucose, and in the liver it is
saturated under normal conditions, and so acts at a constant rate to provide G-6-p
to meet the liver’s needs. Liver cells also contain an isoenzyme of hexokinase,
glucokinase, which has a Km very much higher than the normal intracellular
concentration of glucose. The function of glucokinase in the liver is to remove
glucose from the hepatic portal blood following a meal, so regulating the
concentration of glucose available to peripheral tissues. This provides more G-6-p
than is required for glycolysis; it is used for glycogen synthesis and lipogenesis.
Glucokinase is also found in pancreatic β-islet cells, where it functions to
detect high concentrations of glucose. As more glucose is phosphorylated by
glucokinase, there is increased glycolysis, leading to increased formation of ATP.
This leads to closure of an ATP-potassium channel, causing membrane
depolarization and opening of a voltage gated calcium channel. The resultant
influx of calcium ions leads to fusion of the insulin secretory granules with the cell
membrane, and the release of insulin.

Dr. Ali A. Kasim/ College of Pharmacy/ University of Baghdad

 G-6-p is an important compound at the junction of several metabolic pathways:
glycolysis, gluconeogenesis, the pentose phosphate pathway, glycogenesis, and
glycogenolysis.
2. The isomerization of G-6-P (aldose) to fructose-6-phosphate (F-6-P)
(ketose) by Phosphohexose isomerase.

3. The phosphorylation of F-6-P to fructose 1,6 bisphosphate (F-1,6-BP) by

phosphofructokinase.

The phosphofructokinase reaction is irreversible under physiological conditions.

Phosphofructokinase has a major role in regulating the rate of glycolysis.
4. The cleavage of F-1,6-BP by aldolase, this yields two three-carbon
molecules: glyceraldehyde-3-phosphate (GA-3-P) and dihydroxyacetone
phosphate (DHAP).

Dr. Ali A. Kasim/ College of Pharmacy/ University of Baghdad

 5. The isomerization of DHAP to a second molecule of GA-3-P by triose
phosphate isomerase.

6. The dehydrogenation and concomitant phosphorylation of GA-3-P to

glycerate-1,3-bisphosphate (1,3-BPGA) by glyceraldehyde-3-phosphate
dehydrogenase.

The substrate GA-3-P initially combines with an -SH group present at the
active site of the enzyme, forming a thiohemiacetal that is oxidized to a thiol ester;
the hydrogens removed in this oxidation are transferred to NAD+. The thiol ester
then undergoes phosphorolysis; inorganic phosphate (Pi) is added, forming 1,3-
bisphospho-glycerate , and the free -SH group.

The toxicity of arsenic is the result of competition of arsenate with inorganic

phosphate (Pi) in this reaction to give 1-arseno-3-phosphoglycerate, which
undergoes spontaneous hydrolysis to 3-phosphoglycerate without forming ATP.
The enzyme is also inhibited by the -SH poison iodoacetate.
The oxidation of glyceraldehyde 3-phosphate GA-3-P and the site of action of
the site of action of toxicant are shown in the following figure.

Dr. Ali A. Kasim/ College of Pharmacy/ University of Baghdad

 7. The phosphoryl group transfer from 1,3-BPGA to ADP by
phosphoglycerate kinase, which yields ATP and 3-phosphoglycerate (3-
PGA).

Since two molecules of triose phosphate are formed per molecule of glucose
undergoing glycolysis, two molecules of ATP are formed in this reaction per
molecule of glucose undergoing glycolysis.
In erythrocytes, the reaction catalyzed by phosphoglycerate kinase may be
bypassed to some extent by the reaction of bisphosphoglycerate mutase,
which catalyzes the conversion of 1,3-bisphosphoglycerate to 2,3-
bisphosphoglycerate, followed by hydrolysis to 3-phosphoglycerate and Pi,
catalyzed by 2,3-bisphosphoglycerate phosphatase; as in the following figure.

Dr. Ali A. Kasim/ College of Pharmacy/ University of Baghdad

 This pathway involves no net yield of ATP from glycolysis, but provides
2,3-bisphosphoglycerate, which binds to hemoglobin, decreasing its affinity for
oxygen, so making oxygen more readily available to tissues.

8. The isomerization of 3-PGA to 2-PGA by phosphoglycerate mutase.

Dr. Ali A. Kasim/ College of Pharmacy/ University of Baghdad

 9. The dehydration of 2-PGA to phosphoenolpyruvate (PEP) by enolase.

Enolase is inhibited by fluoride, and when blood samples are taken for
measurement of glucose, glycolysis is inhibited by taking the sample into tubes
containing fluoride.
10.The transfer of the phosphoryl group from PEP to ADP by pyruvate
kinase, to yield a second molecule of ATP.

The reaction of pyruvate kinase is essentially irreversible under physiological

conditions,

NOTE:
The entire glycolysis pathway can be separated into two phases:
- The Preparatory phase (Reactions 1-5) – in which ATP is consumed (in
reactions 1 and 3); hence is also known as the investment phase.
- The Pay Off phase (Reactions 6-10)– in which ATP is produced (in
reactions 7 and 10).
The overall process of glycolysis is:
Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP

Dr. Ali A. Kasim/ College of Pharmacy/ University of Baghdad

The Fates of Pyruvate
In terms of energy, the result of glycolysis is the production of two ATPs
and two NADHs per molecule of glucose.
Two molecules of ATP are expended in the initial phosphorylation steps (1 and 3).
ATP is gained in steps 7 and 10 (these two steps are called substrate-level
phosphorylation steps). Since all of the steps from 6 to 10 occur twice per
molecule of glucose, the net balance is a gain of two moles of ATP per mole of
glucose – a very modest number indeed (considering that the overall yield in
complete oxidative degradation is around 30 moles of ATP).
Pyruvate, the other product of glycolysis, is still an energy-rich molecule, which
can yield a substantial amount of ATP. Whether or not further energy can be
produced, however, depends on the availability of oxygen and the cell type.
Under aerobic conditions, most cells in the body convert pyruvate into
acetyl-CoA by pyruvate dehydrogenase complex, which requires thiamin
diphosphate (Vitamin B1) as a coenzyme.

Note: this reaction occurs in the

mitochondria; pyruvate enters the
mitochondria via a proton
symporter.

Vitamin B1 deficiency is associated with impairment of glucose metabolism,

with significant (and potentially life-threatening) lactic and pyruvic acidosis.
Pyruvate, formed in the cytosol, is transported into the mitochondrion by a
proton symporter. Inside the mitochondrion, it is oxidatively decarboxylated to
acetyl-CoA by a multienzyme complex that is associated with the inner
mitochondrial membrane.
This pyruvate dehydrogenase complex is analogous to the α-ketoglutarate
dehydrogenase complex of the citric acid cycle.

pyruvate dehydrogenase complex is composed of three subunits with different

enzymatic actions; that are:

Dr. Ali A. Kasim/ College of Pharmacy/ University of Baghdad

 1. Pyruvate dehydrogenase which act to decarboxylate pyruvate and forming
a hydroxyethyl derivative of the thiazole ring of enzyme-bound thiamin
diphosphate.
Thiamin is vitamin B1 and in deficiency, glucose metabolism is impaired,
and there is significant (and potentially life-threatening) lactic and pyruvic
acidosis.
2. Dihydrolipoyl transacetylase that contain oxidized lipoamide as a
prosthetic group. Thiamin diphosphate reacts with the oxidized lipoamide
to form acetyl lipoamide, which in turn react with coenzyme A to form
acetyl-CoA and reduced lipoamide.
Note: this subunit forms a long flexible arm, allowing the lipoic acid
prosthetic group to rotate sequentially between the active sites of each of the
enzymes.
3. Dihydrolipoyl dehydrogenase which is a flavoprotein; i.e. containing FAD
as prosthetic group.
Dihydrolipoyl dehydrogenase reoxidzes the reduced lipoamide by FAD,
which is reduced into FADH2. FADH2 in turn is reoxized by NAD+ into
FAD and forming NADH. The reducing equivalents is transfered to the
respiratory chain.

The following figure is schematic representation of the pyruvate

dehydrogenase complex structure and actions.

Dr. Ali A. Kasim/ College of Pharmacy/ University of Baghdad

 The overall reaction is:
Pyruvate + NAD+ + CoA → Acetyl-CoA + NADH + H+ + CO2

The pyruvate dehydrogenase complex consists of a number of polypeptide

chains of each of the three component enzymes, and the intermediates do not
dissociate, but are channeled from one enzyme site to the next. This increases the
rate of reaction and prevents side reactions, increasing overall efficiency.

Dr. Ali A. Kasim/ College of Pharmacy/ University of Baghdad

Under anaerobic condition pyruvate is reduced by lactate dehydrogenase to
lactate

Note: this reaction occurs in the cytosol

This is true of skeletal muscle, where the rate of work output, and hence the
need for ATP formation, may exceed the rate at which oxygen can be taken up and
utilized.
Glycolysis in erythrocytes always terminates in lactate, because the subsequent
reactions of pyruvate oxidation are mitochondrial, and erythrocytes lack
mitochondria.
To make oxygen-free (anaerobic) glycolysis feasible, we have to solve one
problem. In the glyceraldehyde-3-phosphate dehydrogenase reaction, a molecule of
NAD+ is consumed and converted to NADH. Under aerobic conditions, that is
when oxygen is available; NADH is reverted to NAD+ in the respiratory chain.
However, under anaerobic conditions, we need another means to regenerate NAD +.
This problem is overcomed by the reduction of pyruvate to lactate.

Dr. Ali A. Kasim/ College of Pharmacy/ University of Baghdad

 The maximal level of exertion of skeletal muscles cannot be kept up for
long. One will soon experience exhaustion or tiredness and pain. Exhaustion is due
to the depletion of ATP and of glucose, and inhibition of glycolysis due to lactate
accumulation which produces acidosis that inhibits the glycolytic enzymes (most
importantly PFK); while pain is due to the accumulation of lactate in the tissues
and the blood.
Other tissues that normally derive much of their energy from glycolysis and
produce lactate include brain, gastrointestinal tract, renal medulla, retina, and skin.
Lactate production is also increased in septic shock, and many cancers also
produce lactate. The liver (mostly), kidneys, and heart normally take up lactate and
oxidize it.
Glycolysis and Pyruvate dehydrogenase Regulation
Although most of the reactions of glycolysis are freely reversible, three are
markedly exergonic and must therefore be considered to be physiologically
irreversible.
These reactions, catalyzed by hexokinase, phosphofructokinase, and pyruvate
kinase, are the major sites of regulation of glycolysis.
Phosphofructokinase is the most important control element in the mammalian
glycolytic pathway. It is significantly inhibited at normal intracellular
concentrations of ATP; this inhibition this inhibition can be rapidly relieved by
5′AMP that is formed as ADP begins to accumulate, signaling the need for an
increased rate of glycolysis. It is also inhibited by citrate.
Fructose and glucose metabolism converge at the level of the triose-phosphates,
and bypasses the main regulatory steps, so resulting in formation of more pyruvate
and acetyl-CoA than is required for ATP formation. In the liver and adipose tissue,
this leads to increased lipogenesis, and a high intake of fructose may be a factor in
the development of obesity.
Pyruvate dehydrogenase is inhibited allosterically by its products, acetyl-
CoA, and NADH. It is also regulated by phosphorylation (catalyzed by a kinase),
resulting in decreased activity and by dephosphorylation (catalyzed by a
phosphatase) that causes an increase in activity. The kinase is activated by
increases in the [ATP]/[ADP], [acetyl-CoA]/[CoA], and [NADH]/[NAD+] ratios.

Dr. Ali A. Kasim/ College of Pharmacy/ University of Baghdad

CLINICAL ASPECTS
Because of the dependence of the brain on glucose as a fuel, these metabolic
defects commonly cause neurological disturbances.
The exercise capacity of patients with muscle phosphofructokinase
deficiency is low, particularly if they are on high-carbohydrate diets. By providing
lipid as an alternative fuel, work capacity is improved, when blood free fatty acid
and ketone bodies are increased.
Inherited pyruvate kinase and aldolase deficiency in erythrocytes cause
hemolytic anemia.
Arsenic and mercuric ions bind with and inhibit pyruvate dehydrogenase, as
does a dietary deficiency of thiamin, allowing pyruvate to accumulate. Many
alcoholics are thiamin deficient (both because of a poor diet and because alcohol
inhibits thiamin absorption), and may develop potentially fatal pyruvic and lactic
acidosis. Patients with inherited pyruvate dehydrogenase deficiency, also present
with lactic acidosis, particularly after a glucose load.

Dr. Ali A. Kasim/ College of Pharmacy/ University of Baghdad