Glycolysis

Glycolysis is a central metabolic pathway responsible for

the breakdown of glucose and plays a vital role in
generating free energy for the cell and metabolites for
further oxidative degradation. Glucose primarily becomes
available in the blood as a result of glycogen breakdown
or from its synthesis from noncarbohydrate precursors
(gluconeogenesis) and is imported into cells by specific
transport proteins. Glycolysis occurs in the cytoplasm
and consists of 10 reactions, the net result of which is the
conversion of 1 C6 glucose to 2 C3 pyruvate molecules.
The free energy of this process is harvested to produce
adenosine triphosphate (ATP) and nicotinamide adenine
dinucleotide hydride (NADH), key energy-yielding
metabolites. The overall stoichiometry of the pathway is:
glucose + 2 Pi + 2 ADP + 2 NAD+ > 2 pyruvate + 2 ATP + 2
NADH + 2 H+ + 2 H2O (H+: hydrogen ion, Pi: phosphate ion,
NAD+: nicotinamide adenine dinucleotide).

Steps 1–5: 1st Half of Glycolysis

The first half of glycolysis requires an energy investment

of 2 adenosine triphosphate (ATP) molecules and serves to convert
the hexose glucose into 2 trioses.
The process consists of 5 steps:

1. Glucose → glucose 6-phosphate (G6P)

o Hexokinase (HK) transfers a phosphoryl group from ATP
onto the 6th carbon of glucose to form G6P.
 Requires magnesium (Mg ) as a cofactor
2+

 Requires ATP
o In the liver, this step is catalyzed by glucokinase (an
enzyme with the same function but lower glucose
affinity), helping the liver serve as a blood glucose
"buffer."
2. G6P → fructose-6-phosphate (F6P)
o Phosphoglucose isomerase (PGI) converts G6P to F6P.
o Isomerizes the aldose glucose to a ketose fructose

3. F6P → fructose-1,6-biphosphate (FBP)

o Phosphofructokinase (PFK-1) phosphorylates F6P on
C1, yielding FBP.
o Requires Mg2+
as a cofactor
o Requires ATP
o This is a rate-determining reaction in glycolysis,
therefore a regulated step

4. FBP → glyceraldehyde 3-phosphate (GAP) +

dihydroxyacetone phosphate (DHAP)
o Aldolase cleaves the 6-carbon FBP into 2 different 3-
carbon molecules, GAP and DHAP.
o The reaction is an aldol cleavage with an enolate
intermediate stabilized by resonance.

5. DHAP → GAP
o Triose-phosphate isomerase (TIM) interconverts DHAP
and GAP to allow DHAP to proceed through glycolysis.
 Steps 6–10: 2nd Half of Glycolysis

The second half of glycolysis converts the triose GAP to

pyruvate, with the concomitant generation of 4 ATP and
2 nicotinamide adenine dinucleotide hydride (NADH) per 2 GAP.
Thus, the energy investment of steps 1–5 is paid back twice here. In
certain cell types and conditions, these 5 steps are the predominant
source of ATP:

6. GAP → 1,3-bisphosphoglycerate (1,3-BPG)

o Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
catalyzes the phosphorylation and oxidation of GAP,
yielding 1,3-biphosphoglycerate (1,3-BPG).
o 1,3-BPG is the 1st high-energy intermediate in
glycolysis.
o Produces 2 NADH from nicotinamide adenine
dinucleotide (NAD ) and a phosphate ion (Pi)
+

 Under aerobic conditions, oxidation of NADH at the

respiratory chain regenerates NAD and produces+

additional ATP.
 Under anaerobic conditions, additional reactions are
required to regenerate NAD . +
7. 1,3-BPG → 3-phosphoglycerate
o Phosphoglycerate kinase (PGK) converts 1,3-BPG to 3-
phosphoglycerate (3PG).
o Requires Mg 2+
as a cofactor
o Produces ATP
o The GAPDH and PGK reactions are coupled to allow the
energetically unfavorable GAPDH reaction to be “pulled
forward” by the highly favorable PGK reaction.

8. 3PG → 2-phosphoglycerate
o Phosphoglycerate mutase (PGM) converts 3PG to 2-
phosphoglycerate (2PG) by transferring the functional
group phosphate from C3 to C2.
o Generates a 2,3-bisphosphoglycerate (2,3-BPG)–enzyme
complex

9. 2PG → phosphoenolpyruvate (PEP)

o Enolase dehydrates 2PG to phosphoenolpyruvate
(PEP).
o PEP is the 2nd high-energy intermediate formed in
glycolysis.

10. PEP → pyruvate

o Pyruvate kinase (PK) converts PEP to pyruvate (Pyr),
releasing a large amount of energy, which is used to
drive the synthesis of ATP.
o Produces ATP

Net reaction: glucose + 2 Pi + 2 ADP + 2 NAD+ → 2 pyruvate +

2 ATP + 2 NADH + 2 H+ + 2 H2O
 Regulation of Glycolysis

 Glycolysis operates continuously in most tissues, with

a varying rate according to the needs of the cell.
 Factors that induce glycolysis
repress gluconeogenesis (the reverse of glycolysis)
and vice versa because gluconeogenesis is
reciprocally regulated.
 Insulin and glucagon are the main hormones that
control the fluxes of glycolysis and gluconeogenesis.
 Optimal pathway regulation is achieved by controlling
reactions with a large negative free energy change, of
which there are 3 in glycolysis.
 An overview of the regulation of glycolysis. Activators
of hexokinase (HK), phosphofructokinase-1 (PFK-1), or
pyruvate kinase (PK) are marked in green. Metabolites
that inhibit these enzymes are marked in red.

Hexokinase (HK)

 Regulates step 1 of the pathway

 Negatively regulated by excess G6P
 Not relevant when glucose is derived from glycogen,
as

Glucose is released from glycogen as G6P

Phosphofructokinase

 PFK-1 is the primary flux control point for glycolysis;

regulates step 3
 FBPase catalyzes the reverse step to PFK-1
in gluconeogenesis, and the 2 enzymes are
reciprocally regulated.
o When PFK-1 is inhibited and FBPase is activated, flux is
shifted from glycolysis to gluconeogenesis.
 PFK-1 is allosterically inhibited by ATP,
an indicator of energy abundance.
 PFK-1 is allosterically activated
by adenosine monophosphate (AMP)
and adenosine diphosphate (ADP), indicators of
energy scarcity.
 PFK-1 is allosterically inhibited by citrate.
 PFK-1 is potently allosterically activated by fructose-
2,6-bisphosphate (F2,6P).
o F2,6P has the opposite effect on the opposing step
in gluconeogenesis.
o F2,6P is synthesized and degraded by a bifunctional
enzyme called PFK-2/FBPase-2, whose activity is
controlled by many allosteric effectors and hormones.
o F6P promotes F2,6P synthesis, activating glycolysis.
o In the fed state: insulin stimulates PFK-2/FBPase-2
dephosphorylation → increasing F2,6P levels →
increasing glycolytic flux
 Catecholamines (via cyclic AMP) inhibit
glycolytic enzymes HK, PFK-1, PFK-2 (which produces
fructose 2,6 bisphosphate), and PK.
o Inducing synthesis of pyruvate carboxylase, PEP
carboxykinase, FBPase, and G6Pase
 Pyruvate kinase (PK)

 Regulates step 10 (last) of the pathway

 Allosterically activated by FBP, indicating accumulation
of upstream glycolytic intermediates: results in
"pulling" through the glycolytic pathway
 Allosterically inhibited by ATP, indicating plentiful
energy supply
 In the liver, allosterically inhibited by alanine, a
precursor for gluconeogenesis

Clinical Relevance

 Galactosemia: defective metabolism of the

sugar galactose. Clinical manifestations begin when
milk feeding is started. Infants
develop lethargy, jaundice,
progressive liver dysfunction, kidney disease,
cataracts, weight loss, and susceptibility to
bacterial infections (especially E coli).
Intellectual disability may develop if the disorder is
left untreated. The mainstay of management is
exclusion of galactose from the diet.
 Hereditary fructose intolerance: deficiency of fructose-
1-phosphate aldolase. Symptoms begin after ingestion
of fructose (fruit sugar) or sucrose so presents later in
life. Presents with failure to gain
weight, vomiting, hypoglycemia, liver dysfunction,
and kidney defects. Children with the disorder do very
well if they avoid dietary fructose and sucrose.
 Fructose 1,6-diphosphatase deficiency: associated
with impaired gluconeogenesis. Symptoms
include hypoglycemia, intolerance to fasting, and
hepatomegaly. Emergent treatment of hypoglycemic
episodes with glucose rich IV fluids and avoidance of
fasting are the mainstays of therapy. Severe cases may
 require glucose supplementation to
avoid hypoglycemia.
 Glycogen storage diseases: deficiency
of enzymes responsible for glycogen degradation.
Depending upon which enzyme is affected, these
conditions may affect the liver, muscles, or both.
There are several clinically significant glycogen
storage diseases with differing presentations.
 Glucose 6-phosphate dehydrogenase deficiency
(G6PD): a genetic disorder that occurs almost
exclusively in males and mainly affects red blood cells,
causing hemolysis and hemolytic anemia. Symptoms
include dyspnea, fatigue, tachycardia, dark urine,
palor, and jaundice. Hemolytic anemia may be
triggered by infections, certain drugs (antibiotics,
antimalarials), and after eating fava beans.

The following are enzymes of the glycolysis pathway that may be

involved in congenital enzymatic defects:

 Pyruvate kinase deficiency (most common)

 Erythrocyte hexokinase
 Glucose phosphate isomerase
 Phosphofructokinase

These congenital enzymatic defects produce hemolytic anemia.

Hemolytic anemia: a group of anemias that are due to destruction
or premature clearance of RBCs. Intrinsic abnormalities of the RBC
lead to splenic clearance (extravascular hemolysis). The chronic
destruction of RBCs can present
as jaundice, splenomegaly, cholelithiasis, hematuria, and symptoms
of anemia
(shortness of breath, fatigue, syncope, and tachycardia).