Reading: Harper's Biochemistry pp. 190-198 Lehninger Principles of Biochemistry 3rd Ed. pp. 527-566

1. GLYCOLYSISGLYCOLYSIS
Reading:
Harper’s Biochemistry pp. 190-198
Lehninger Principles of Biochemistry
3rd Ed. pp. 527-566
Glycolysis- from the Greek-
glykys- “sweet”
lysis- “splitting”

2. OBJECTIVESOBJECTIVES
To understand how the glycolytic pathway is used
to convert glucose to pyruvate (and lactate) with
conservation of chemical potential energy in the
form of ATP and NADH.
To learn the intermediates, enzymes, and cofactors
of the glycolytic pathway.

3. Major pathways of glucose utilization inMajor pathways of glucose utilization in
cells of higher plants and animalscells of higher plants and animals
Although not the only
possible fates for
glucose, these three
pathways are the
most significant in
terms of the amount
of glucose that flows
through them in most
cells.

4. Glucose is the major fuel of most organisms.
It is relatively rich in potential energy- complete
oxidation to CO2 and H2O proceeds with a free-
energy change of -2,840 kJ/mol.
By storing glucose as high molecular weight
polymers (starch/glycogen) a cell can stockpile
large quantities of hexose units.
When energy demands increase, glucose can be
released quickly from storage and used to produce
ATP either aerobically or anaerobically.

5. Glycolysis occurs in the cytosol of cells, is common
to most organisms, and in humans occurs in
virtually all tissues.
Most tissues have at least a minimal requirement
for glucose. In the brain, the requirement for
glucose is substantial, in erythrocytes, it is nearly
total.
In glycolysis, a molecule of glucose is degraded in a
series of steps catalyzed by ten cytosolic enzymes,
to yield two molecules of the 3 carbon compound,
pyruvate.
During those sequential reactions, some of the free
energy released is conserved in the form of ATP
and NADH

6. Biomedical ImportanceBiomedical Importance
Of crucial biomedical significance is the ability of glycolysis to
provide ATP in the absence of oxygen.
This allows skeletal muscle to perform at high levels when aerobic
oxidation becomes insufficient, and allows cells to survive anoxic
episodes.
Diseases associated with impaired glycolysis:
♦Hemolytic anemia:
- of the defects in glycolysis that cause hemolytic anemia, pyruvate
kinase deficiency (genetic mutations) is the most common.
- mature erythrocytes contain no mitochondria, totally dependent
upon glycolysis for ATP.
- ATP is required for Na/K-ATPase-ion transport system which
maintain the proper shape of the erythrocyte membrane.
♦Lactic Acidosis:
- can be due to several causes of improper utilization of lactate.

7. Glycolysis is only the first step in theGlycolysis is only the first step in the
degradation of glucosedegradation of glucose
Three possible
catabolic fates of the
pyruvate formed in
glycolysis. Pyruvate
also serves as a
precursor in many
anabolic reactions, not
shown here.

8. Where glycolysis fits inWhere glycolysis fits in
the big picture ofthe big picture of
catabolismcatabolism

9. Overview ofOverview of
glycolysisglycolysis

12. Energy Transformations during GlycolysisEnergy Transformations during Glycolysis
Glucose + 2 NAD+
+ 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+
+ 2 ATP + H2O
(Note: Much, 95%, of the energy remains in pyruvate)
Resolve into two processes:
Glucose + 2 NAD+
→ 2 pyruvate + 2 NADH + 2 H+
∆Go´
1 = -146 kJ/mol
2 ADP + 2 Pi → 2 ATP + 2 H2O
∆Go´
= 2(30.5 kJ/mol) = 61 kJ/mol
Overall free energy change = -146 + 61 = -85 kJ/mol

13. Why phosphorylated intermediates?Why phosphorylated intermediates?
Each of the nine glycolytic intermediates between glucose and
pyruvate is phosphorylated
1. Phosphate groups are ionized at pH 7, giving each
glycolytic intermediate a net negative charge. Because the
plasma membrane is impermeable to charged molecules, the
phosphorylated intermediates cannot disperse out of the cell.
2. Energy used in the formation of the phosphate ester is
partially conserved. High energy phosphate compounds
formed in glycolysis (1,3-bisphosphoglycerate and
phosphoenolpyruvate) donate phosphoryl groups to ADP to
form ATP.
3. Binding energy resulting from the binding of phosphate
groups to the active sites of enzymes lowers the activation
energy and increases the specificity of the enzymatic
reactions.

14. Step 1: Phosphorylation of glucoseStep 1: Phosphorylation of glucose
Glucose is activated by phosphorylation at C6
Reaction is catalyzed by hexokinase, present in virtually all
extrahepatic cells. Has high affinity (low Km) for glucose, so
phosphorylates essentially all the glucose that enters cell,
maintaining a large glucose gradient. Will also phosphorylate other
hexose sugars. Under physiological conditions, reaction is
essentially irreversible.
In liver, glucose is phosphorylated by glucokinase. This enzyme has
a low affinity (high Km) for glucose, is specific for glucose, and its

15. Step 2: Conversion of glucose 6-phosphateStep 2: Conversion of glucose 6-phosphate
to fructose 6-phosphateto fructose 6-phosphate
The enzyme phosphohexose isomerase catalyzes
the reversible isomerization of glucose 6-phosphate
(an aldose) to fructose 6-phosphate (a ketose)
As predicted for the relatively small change in
standard free energy, the reaction proceeds readily
in either direction, and requires Mg2+

16. Step 3: Phosphorylation of fructose 6-Step 3: Phosphorylation of fructose 6-
phosphate to fructose 1, 6-bisphosphatephosphate to fructose 1, 6-bisphosphate
Phosphofructokinase-1 catalyzes the transfer of a phosphoryl
group from ATP to fructose 6-phosphate to yield fructose 1, 6-
bisphosphate.
This reaction is essentially irreversible under cellular conditions.
Phosphofructokinase-1 is a regulated enzyme at a major point in
the regulation of glycolysis.
PFK-1 activity is increased whenever the cell’s ATP supply is
depleted or when ADP/Pi are in excess.
Activity is inhibited whenever the cell has ample ATP and is well
supplied by other fuels such as fatty acids.

17. Step 4: Cleavage of fructose 1, 6-Step 4: Cleavage of fructose 1, 6-
bisphosphatebisphosphate
The enzyme fructose 1, 6-bisphosphate aldolase,
called just aldolase, catalyzes the cleavage of
fructose 1, 6-bisphosphate into two different triose
phosphate, glyceraldehyde 3-phosphate and
dihydroxyacetone phosphate
In cells, this reaction can proceed in either direction,
and proceeds to the right during glycolysis because
products are quickly removed.

18. Step 5: Interconverstion of the trioseStep 5: Interconverstion of the triose
phosphatesphosphates
Dihydroxyacetone phosphate is rapidly and reversibly converted
to glyceraldehyde 3-phosphate by triose phosphate isomerase
The C-1, C-2, C-3 of the starting glucose now become chemically
indistinguishable for the C-6, C-5, and C-4, respectively.
This reaction completes the prepatory phase of glycolysis
Other hexoses (fructose, mannose, galactose) can also be

19. The payoff phase of glycolysis producingThe payoff phase of glycolysis producing
ATP + NADHATP + NADH
2 molecules of glyceraldehyde 3-phosphate → 2 molecules of pyruvate
Step 6: Glyceraldehyde 3-phosphate dehydrogenase catalyzes
the oxidation of glyceraldehyde 3-phosphate to 1, 3-
bisphosphoglycerate
Note that the aldehyde group is dehydrogenated to an acyl
phosphate, which has a very high standard free energy of
hydrolysis (-49.3 kJ/mol).
Glyceraldehyde 3-phosphate dehydrogenase is inhibited by
iodoacetetate

20. Step 7: Phosphoryl transfer from 1, 3-Step 7: Phosphoryl transfer from 1, 3-
bisphospho-glycerate to ADPbisphospho-glycerate to ADP
The enzyme phosphoglycerate kinase transfers the high-energy
phosphoryl group from the carboxyl group to ADP, forming ATP
and 3-phosphoglycerate.
Steps 6 and 7 represent an energy-coupling process in which 1, 3-
phosphoglycerate is the common intermediate.
Glyceraldehyde 3-phosphate + ADP + Pi +NAD+
3-phosphoglycerate + ATP + NADH + H+
∆Go´
1= -12.5 kJ/mol

21. Step 8: Conversion of 3-phosphoglycerateStep 8: Conversion of 3-phosphoglycerate
to 2-phosphoglycerateto 2-phosphoglycerate
The enzyme phosphoglycerate mutase catalyzes a
reversible shift of the phosphoryl group between C-
2 and C-3 of glycerate. Mg2+
is essential

22. Step 9: Dehydration of 2-phosphoglycerateStep 9: Dehydration of 2-phosphoglycerate
to phosphoenolpyruvateto phosphoenolpyruvate
Enolase promotes reversible removal of a molecule of
water from 2-phosphoglycerate to yield
phosphoenolpyruvate
Standard free energy of hydrolysis of the phosphate
groups of the reactant and product are -17.6 kJ/mol and
-61.9 kJ/mol, respectively.
ie. The loss of the water molecule causes a
redistribution of energy within the molecule, generating
a super high-energy phosphate compound.

23. Step 10: Transfer of the phosphoryl groupStep 10: Transfer of the phosphoryl group
from phosphoenolpyruvate to ADPfrom phosphoenolpyruvate to ADP
This last step in glycolysis is catalyzed by pyruvate kinase,
which requires K+
and Mg 2+
or Mn 2+
This step is also an important site of regulation
The product pyruvate undergoes tautomerization from its
enol to keto form which is more stable at pH 7

24. Overall balance sheet - net gain of ATPOverall balance sheet - net gain of ATP
Glucose + 2 ATP+ 2 NAD+
+ 4 ADP + 2 Pi →
2 Pyruvate + 2 ADP + 2 NADH + 2 H+
+ 4 ATP + 2 H2O
or Glucose + 2 NAD+
+ 2 ADP + 2 Pi →
2 Pyruvate + 2 NADH + 2 H+
+ 2 ATP + 2 H2O
Under aerobic conditions, the two molecules of NADH are reoxidized
to NAD+
by transfer of their electrons to the respiratory chain in the
mitochondrion
2 NADH + 2 H+
+ O2 → 2 NAD+
+ 2 H2O
During glycolysis:
*Carbon pathway - Glucose → 2x pyruvate
*Phosphate pathway - 2 ADP + 2 Pi → 2 ATP
*Electron pathway - Four electrons (two hydride ions) are
transferred from 2 molecules of glyceraldehyde
3-phosphate to two of NAD+

25. Conversion of pyruvate to lactateConversion of pyruvate to lactate
Under hypoxic or anaerobic
conditions, NADH
generated by glycolysis
cannot be reoxidized by O2 -
NAD+
is required during
glycolysis as electron
acceptor in step 6.
In these cases, NAD+
is
regenerated from NADH by
the reduction of pyruvate to
lactate, catalyzed by lactate
dehydrogenase. This
allows glycolysis to occur
in the absence of oxygen
Lactate produced in muscle
during a short burst of
physical activity is
converted back to glucose
in the liver.

26. Glycolysis is regulated at 3 steps involvingGlycolysis is regulated at 3 steps involving
non equilibrium reactionsnon equilibrium reactions
Step 1: hexokinase
glucose → glucose 6-phosphate
Step 3: phosphofructokinase
fructose 6-phosphate → fructose 1, 6-bisphosphate
Step 10: pyruvate kinase
phosphoenolpyruvate → pyruvate
These are all exergonic and physiologically irreversible
These enzymes function as “valves”, regulating the flow of carbon through
glycolysis. The rates of these steps are limited not by the substrate but by the
activity of the enzymes.
Enzymes that catalyze these exergonic, rate-limiting steps are commonly the targets
of metabolic regulation.
Examples of regulation:
Phosphofructokinase-1 - inhibited by high levels of ATP. ATP binds to an allosteric
site and lowers affinity for fructose 6-phosphate
Hexokinase - allosterically inhibited by its product.
Pyruvate kinase - inhibited by ATP

27. Regulation occurs at steps that
are enzyme-limited. At each of
these steps (orange arrows),
which are generally exergonic, the
substrate is not in equilibrium
with the product because the
reaction is relatively slow; the
substrate tends to accumulate,
just as river water accumulates
behind a dam. In the substrate-
limited reactions (blue arrows),
the substrate and product are
essentially at their equilibrium
concentrations. In the steady
state, all reactions in the
sequence occur at the same rate,
which is determined by the rate-
limiting step.
Regulation of the flux through a multistepRegulation of the flux through a multistep
pathwaypathway

28. Feeder pathways for glycolysisFeeder pathways for glycolysis

29. SUMMARYSUMMARY
Glycolysis is a universal metabolic pathway for the
catabolism of glucose to pyruvate accompanied by the
formation of ATP.
The process is catalyzed by 10 cytosolic enzymes and
there is a net gain of two ATPs per molecule of glucose.
The NADH formed must be recycled to regenerate NAD+
.
Under aerobic conditions this occurs during mitochondrial
respiration; under anaerobic conditions, NAD+
is
regenerated by the conversion of pyruvate to lactate.
Other organisms such as yeast regenerate NAD+
by
reducing pyruvate to ethanol + CO2 (fermentation)
A variety of D-hexoses, including fructose, mannose, and
galactose, can be funneled into glycolysis.
Enzyme limited, regulated steps are catalyzed by
hexokinase, phosphofructokinase-1, and pyruvate kinase.