BIOCHEMISTRY
1
LECTURE: GLYCOLYSIS AND THE OXIDATION OF PYRUVATE
LECTURER: Josephine B. Guerrero, MD, FPCGM
OUTLINE
I. Biomedical Importance
II. The Reactions of Glycolysis Constitute the Main
Pathway of Glucose Utilization
III. Summary of Glycolysis
IV. How does Glucose Enter the Cell?
V. Tissues That Function Under Hypoxic Conditions
Produce Lactate
VI. Three Steps Involving Nonequilibrium Reactions
VII. The Oxidation of Pyruvate to Acetyl-Coa Is the
Irreversible Route from Glycolysis To The Citric
Acid Cycle
VIII. Inhibition of Pyruvate Metabolism Leads to Lactic
Acidosis
LEARNING OBJECTIVES
• Identify the pathway for glucose metabolism
• Describe the pathway of glycolysis
• Identify the regulatory mechanisms for glycolysis
• Discuss how energy is produced from glycolysis
• Identify the end product of glycolysis and its fate
• Describe the pyruvate dehydrogenase complex
REFERENCES
• Dr. Guerrero’s PPT
 Ferrier DR, Harvey RA (ed.). 2014. Lippincott’s Illustrated Reviews:
Biochemistry, 6/e. Lippincott WIlliams & Wilkins
 Lieberman M, Marks AD, Peet A. 2013. Marks’ Basic Medical
Biochemistry - A Clinical Approach, 4/e. Lippincott Williams &
Wilkins
 Nelson DL, Cox MA. 2013. Lehninger Principles of Biochemistry,
13/e. W.H. Freeman and Company
 Rodwell VW, Bender DA, Botham KM, Kennelly PJ, Weil PA. 2016.
Harper’s Illustrated Biochemistry, 30/e. McGraw Hill Education
 Rosenthal MD, Glew RH. 2009. Medical Biochemistry - Human
Metabolism in Health and Disease. John Wiley & Sons, Inc.
I. BIOMEDICAL IMPORTANCE
GLYCOLYSIS
 Also called the Embden-Meyerhof pathway
• Main pathway for carbohydrate (glucose) metabolism
• Site: Cytosol of all cells
• Substrate: Glucose (6C)
• Product: Pyruvate (3C) fates:
o Aerobic conditions: oxidized to CO2 and H2O
 Requires both oxygen and mitochondrial
enzyme systems:
▪ Pyruvate dehydrogenase complex
▪ Citric acid cycle
▪ Respiratory chain
o Anaerobic conditions: reduced to lactate
 NOT ALL THE STEPS OF GLYCOLYSIS ARE
EXERGONIC. Some are endergonic while others are
exergonic. However, the overall ∆G of glycolysis is
Lecture Title: Glycolysis and the Oxidation of Pyruvate
Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA
3.1.
negative, making the entire process exergonic and
thus proceeds spontaneously (overall ∆G < 0)
 Erythrocytes - lacks mitochondria  completely reliant
on glucose as their metabolic fuel
 Tissues of the brain – contains mitochondria but
reliant on glycolysis since fatty acids cannot cross the
blood-brain barrier.
o The brain CANNOT utilize β-oxidation
o The brain can meet no more than about 20%
of its energy needs from ketone bodies
• Regulated at three steps:
o Hexokinase
o Phosphofructokinase-1 [Main regulatory
enzyme]
o Pyruvate kinase
 Anaerobic glycolysis provides ATP in the absence of
oxygen – allows:
o Skeletal muscle to perform at very high levels
of work output
o Survival from anoxic episodes
 Heart muscle – adapted for aerobic performance:
o Relatively low glycolytic activity
o Poor survival under conditions of ischemia
 Hemolytic anemias – disease mainly seen in glycolysis
enzyme deficiency (e.g. pyruvate kinase)
 Fatigue – enzyme defect that affects the skeletal
muscle (e.g. phosphofructokinase)
 Proceeds at high rate in fast-growing cancer cells 
large amounts of pyruvate formed  reduced to lactate
and exported  acidic local environment in the tumor
 implications for cancer therapy
The Warburg Effect
Cancer cells consume glucose at a much higher rate and produce
more lactic acid than their normal counterparts, even under aerobic
conditions. A large fraction of the increased ATP produced by
glycolysis in cancer cells is used for fatty acid, protein, and DNA
synthesis, all three of which are increased in cancer cells. The
Warburg effect also provides tumors with large amounts of lactate
and pyruvate that are precursors to the acetyl-CoA substrate that
fatty acid synthesis requires.
 Lactate is used for gluconeogenesis (liver), which is
responsible for hypermetabolism seen in cancer
cachexia
 Lactic acidosis results from:
o Impaired pyruvate dehydrogenase activity
o Thiamin (vit. B1) deficiency
II. THE REACTIONS OF GLYCOLYSIS CONSTITUTE
THE MAIN PATHWAY OF GLUCOSE UTILIZATION
 The overall equation for glycolysis from glucose to
pyruvate is as follows:
Glucose + 2 ATP + 2 NAD+ + 4 ADP + 2 Pi
↓
2 pyruvate + 2 ADP + 2 NADH + 2 H+ + 4 ATP +
2 H 2O
• All of the enzymes of glycolysis are cytosolic.
1
 Figure 1: Steps involved in glycolysis.

 Glycolysis occurs in two phases:
o Preparatory phase – phosphorylation of
glucose and its conversion to glyceraldehyde-
3-phosphate
o Payoff phase – oxidative conversion of
glyceraldehyde-3-phosphate to pyruvate and
the coupled formation of ATP and NADH
Step 1: Phosphorylation of Glucose to Glucose 6-
Phosphate
• In the Liver, it removes glucose from the hepatic portal
blood following a meal which regulates the
concentration of glucose available to peripheral tissues.
• It is also found in pancreatic B-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.
Table 1: Hexokinase vs Glucokinase

HEXOKINASE GLUCOKINASE
o Most tissues o Liver parenchymal
o Can phosphorylate cells, B cells of
other hexoses pancreas
o Inhibited by Glu-6-P o Hexokinase D or type
o Low Km (high affinity) IV (same specificity
for glucose, Low as hexokinase)
Vmax o Indirectly inhibited by
Fructose 6- P and
stimulated by glucose
• Glucose enters glycolysis by phosphorylation to o Higher Km, High
glucose 6-phosphate, catalyzed by hexokinase. Vmax
o Glucose sensor
 Phosphorylation of glucose at C-6 important for blood
glucose homeostasis
 Requires Mg2+ as a cofactor o Implicated in MODY 2
• It uses ATP as the phosphate donor. (Maturity Onset
• Traps glucose in the cell. Diabetes in the Young
• The phosphorylation is regarded as irreversible. 2)

• Insulin, controls the transport of glucose

• available for glycolysis in tissues other than the liver.
• Glucokinase, is an isozyme of hexokinase.
Glucokinase has a Km very much higher than the normal
intracellular concentration of glucose.
Lecture Title: Glycolysis and the oxidation of pyruvate 2
Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA

Figure 2: Relative Km activities of hexokinase and glucokinase.
Step 2: Isomerization of Glucose 6-Phosphate to
Fructose 6-Phosphate
• The enzyme phosphohexose isomerase
(phosphoglucose isomerase) catalyzes the reversible
isomerization of glucose 6-phosphate, an aldose, to
fructose-6-phosphate, a ketose.
▪ Reversible
▪ Aldose ----------> Ketose
▪ Catalyzed by phosphohexose isomerase or
phosphoglucose iosomerase
 Requires Mg2+ as a cofactor
Observe how these 2 have the same molecular formula but
different structures. This process is REVERSIBLE but due to low
F6P concentration, reaction usually goes forward (remember
when product < substrate, reaction goes forward).
Isomerization is necessary in preparation for step 4. Remember
that isomers are of the same molecular formula but diff chemical
structure and properties (ex. kinetic properties)
Step 3: Phosphorylation of Fructose 6-Phosphate
Lecture Title: Glycolysis and the oxidation of pyruvate
Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA
 Phosphofructokinase-1 (PFK-1) catalyzes the
transfer of a phosphoryl group from ATP to fructose 6-
phosphate to yield fructose 1,6-bisphosphate.
• Irreversible
▪ This is because the reaction catalyzed by
Phosphofructokinase is coupled hydrolysis of ATP
▪ MOST IMPORTANT CONTROL POINT of
glycolysis;
▪ Rate-limiting step and committed step in
glycolysis.
▪ Catalyzed by PFK-1
▪ Regulated by intracellular levels of ATP and
Fructose 2,6-Bisphosphate (F 2,6-BP)
▪ Requires Mg2+ as a cofactor
Regulation of glycolysis will be discussed in the next section
 Phosphofructokinase is both inducible and subject to
allosteric regulation.
 It has a major role in regulating the rate of glycolysis.
• In the fed state, fructose 2-6-bisphosphate signals
abundance of glucose = increase in glycolysis
IMPORTANT: PFK-1 is inhibited by elevated levels of ATP
(EXCEPT in the liver) and Citrate (from TCA cycle) and is
stimulated by elevated levels of AMP
• Citrate inhibition favors the use of glucose for
glycogen synthesis.
Step 4: Cleavage of Fructose 1,6-Bisphosphate
 Aldolase catalyzes a reversible aldol condensation.
Fructose 1,6-bisphosphate is cleaved to yield two
different triose phosphates, glyceraldehyde 3-
phosphate (G3P), an aldose, and dihydroxyacetone
phosphate (DHAP), a ketose
o the lysis part of glycolysis
• Reversible, not regulated
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 Step 5: Isomerization of Dihydroxyacetone phosphate
(DHAP)
• Interconverts DHAP to G3P
• Catalyzed by triose phosphate isomerase
• Results in the net production of two molecules of
glyceraldehyde 3-phosphate from fructose 1,6-
bisphosphate
• From this point onwards, reactions products will be
twice in number since there are 2 reacting isomers.
 One molecule of glucose yields two molecules of G3P,
and both halves of the glucose molecule follow the
same pathway in the second phase of glycolysis.
Review
Where does glycolysis occur?
_____________________________________________
What is the overall reaction of glycolysis?
_____________________________________________
What are the two steps that need the use of ATP?
_____________________________________________
_____________________________________________
DHAP and G3P are ____________ of each other (be specific as to
what type of isomers!)
What cofactor is needed in a few steps of glycolysis? What are these
steps (so far)? Just list the enzymes catalyzing the steps.
The cofactor is _______
_____________________________________________
What is the main enzyme that is regulated?
_____________________________________________
How is the main enzyme upregulated. There are three:
_______________; _______________; ________________
How is the main enzyme downregulated. There are three:
_______________; _______________; ________________
If oxidation of G3P to 1,3-BPG enters the malate shuttle, how many
ATP is produced? _______
How about the glycerophosphate shuttle? _______
Lecture Title: Glycolysis and the oxidation of pyruvate
Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA
Step 6: Oxidation of Glyceraldehyde-3-Phosphate
 Oxidation of G3P to 1,3-bisphosphoglycerate (1,3-
BPG), catalyzed by glyceraldehyde 3-phosphate
dehydrogenase
▪ First oxidation-reaction of glycolysis
▪ Catalyzed by glyceraldehyde 3-phosphate
dehydrogenase (G3PD)
▪ Coupled to the attachment of a Pi to the carboxyl
group leading to the formation of 1,3-
Bisphosphoglycerate
 Glyceraldehyde-3-phosphate dehydrogenase is NAD+
dependent.
 Enzyme is part of the G3P shuttle
 Structurally, it consists of four identical polypeptides
(monomers) forming a tetramer.
 Four –SH groups are present on each polypeptide,
derived from cysteine residues.
• This step yields 1 NADH
▪ A net of 2 NADH (1 NADH x 2= 2, Remember
product is x2 from this point onwards)
 The NADH produced in this step can enter the
mitochondria either through the malate shuttle or the
glycerophosphate shuttle.
o Malate shuttle – NADH produces 2.5 ATP
o Glycerophosphate shuttle – NADH produces
only 1.5 ATP
Recall in the ETC trans that Glycerophosphate shuttle is the
primary shuttle that FADH2 uses. In this step of glycolysis, NADH
can enter the ETC via the said shuttle producing only 1.5 ATP
since it skips complex I 😊.
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 Step 7: Synthesis of 3-Phosphoglycerate

 The enzyme phosphoglycerate kinase transfers the

high-energy phosphoryl group from the carboxyl group
Figure 3: The 2,3- BPG pathway in erythrocytes.
of 1,3-BPG to ADP, forming ATP and 3-
phosphoglycerate
▪ Physiologically reversible
 The net effect of arsenic on glycolysis is to bypass the
substrate-level phosphorylation catalyzed by
▪ Synthesizes ATP* from the high-energy phosphate phosphoglycerate kinase, reducing the direct net
group energy yield from glycolysis to zero.
▪ Catalyzed by Phosphoglycerate kinase o Direct net energy yield is 0 because 2 ATP is used
▪ Requires Mg2+ as a cofactor up in the first 5 steps and 2 ATP is generated in the
• Substrate-level phosphorylation: the energy needed last 5 steps in the presence of arsenic poisoning.
for the production of a high energy phosphate comes
from a substrate rather than from the electron
transport chain
• 2 ATP (1 ATP x 2 = 2, Remember again, product
x2)

In other words, substrate-level phosphorylation is the generation of

ATP outside the electron transport chain (i.e., not synthesized by
ATP synthase or complex V)

2,3 – BPG and Arsenic Poisoning

 In erythrocytes, the reaction catalyzed by

phosphyoglycerate kinase may be bypassed by the
reaction of bisphosphoglycerate mutase, which
catalyzes the conversion of 1,3-BPG to 2,3-BPG,
followed by hydrolysis to 3-phosphoglycerate and Pi
catalyzed by 2,3-bisphosphoglycerate phosphatase Figure 4: The presence of arsenic skips the step catalyzed by
phosphoglycerate kinase. Because of this, no ATP is produced during this
(Figure 3). step.
o No ATP is synthesized
o 2,3-BPG causes a right shift in the oxyhemoglobin
curve, decreasing its affinity for O2 and allowing
RBCs to deliver more oxygen to tissues
Lecture Title: Glycolysis and the oxidation of pyruvate 5
Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA
 Step 8: Shift of Phosphate group from C3 to C2
 The enzyme phosphoglycerate mutase catalyzes a
reversible shift of the phosphoryl group between C-2
and C-3 of glycerate
▪ Freely reversible
▪ Catalyzed by phosphoglycerate mutase
▪ Requires Mg2+ as a cofactor
Step 9: Dehydration of 2- Phosphoglycerate
 Generates a compound with high phosphoryl group
transfer potential (the first was step 6), enolase
promotes reversible removal of a molecule of water
from 2-phosphoglycerate to yield
phosphoenolpyruvate (PEP).
▪ Reversible
▪ Catalyzed by enolase
▪ Dehydration process
▪ Phosphorylation process
▪ The mechanism of the enolase reaction involves an
enolic intermediate stabilized by Mg2+.
PHOSPHOENOLPYRUVATE is the highest energy compound
of the cell (pinakamalaking negative ΔG, negative syempre)
REVIEW
Briefly define, what is substrate-level phosphorylation?
_________________________________________________
What enzymes catalyze the steps involve in substrate-level
phosphorylation?
_________________________________________________
_________________________________________________
How many ATP is directly produced in glycolysis (the total ATP, not
the net)? Explain why there is a net production of two ATP
molecules if only two ATP is produced per unit of G3P.
___________ ATP/s are produced
_________________________________________________
At the end of glycolysis how many net ATP is produced per unit of
G3P? ________ How about per unit of glucose? ________
Lecture Title: Glycolysis and the oxidation of pyruvate
Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA
Step 10: Formation of pyruvate
 Last step of glycolysis is the transfer of the phosphoryl
group from phosphoenolpyruvate to ADP, catalyzed
by pyruvate kinase producing pyruvate and ATP.
• Third irreversible reaction of glycolysis
• Catalyzed by Pyruvate kinase
• The phosphate of Phosphoenolpyruvate is
transferred to ADP [ADP+ Pi  ATP] catalyzed by
Pyruvate Kinase. Hence, another ATP producing
reaction.
• Pyruvate is produced.
• Process is IRREVERSIBLE.
 Another substrate-level phosphorylation.
 Needed Cofactor: Mg2+
 Requires K+, and either Mg2+ and Mn2+ as
cofactors
III. SUMMARY OF GLYCOLYSIS
 Anaerobic conditions: the NADH cannot be re-
oxidized through the respiratory chain, and pyruvate is
reduced to lactate catalyzed by lactate dehydrogenase.
o This permits the oxidation of NADH to NAD+,
permitting another molecule of glucose to undergo
glycolysis.
 Aerobic conditions: pyruvate is transported into
mitochondria and undergoes oxidative decarboxylation
to acetyl-coA then oxidation to CO2 in the citric acid
cycle.
o The reducing equivalents from NADH formed in
glycolysis are taken up into the mitochondria for
oxidation via: malate-aspartate shuttle or
glycerophosphate shuttle.
▪ oxygen is admitted (aerobic recovery)
 Muscle contraction under aerobic conditions:
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 o Lactate does not accumulate
o Pyruvate is the major end product of glycolysis 
oxidized further to CO2 and H2O

Figure 6: The Cori Cycle

IV. HOW DOES GLUCOSE ENTER THE CELL?

Figure 5: Summary of glycolysis. (-) blocked under anaerobic conditions or
by absence of mitochondria containing key respiratory enzymes, as in
erythrocytes
 Short supply of O2:
oImpaired mitochondrial reoxidation of NADH 
NADH is reoxidized by reducing pyruvate to
lactate  glycolysis continues
 glycolysis under anaerobic conditions limits the amount
of ATP formed per mole of glucose oxidized  much
more glucose must be metabolized
 in yeast, pyruvate formed in anaerobic glycolysis is
decarboxylated and reduced to ethanol
The Cori Cycle
 Family of glucose transporters (GLUT 1 through
GLUT 5) facilitates movement of glucose across the
plasma membrane.
 GLUT 1 – 5 are facilitative bidirectional glucose
transporters (Table 2)
 GLUT 4 – responds to insulin, seen in muscle and
adipocytes. GLUT 4 translocates to and fuses with the
plasma membrane, thus providing the mechanism by
which insulin stimulates uptake of glucose from the
blood (Figure 7).
 GLUT 2 – present in hepatocytes, has a lower affinity
for glucose than does GLUT 4, but is expressed in such
abundance that intracellular glucose equilibrates
essentially instantaneously with glucose in the blood. It
is insulin-independent.
 GLUT 1 and GLUT 3 – red blood cells, brain and
kidney. Like GLUT 2, they are both insulin-independent.
GLUT 1 and GLUT 3 has a high affinity for glucose,
promoting glucose uptake in the fasted state.
 GLUT 5 – expressed in high levels in the small intestine,
where it functions primarily as a transporter of fructose
than of glucose
Table 2: Glucose Transporter Protein Family

 When glycolysis produces lactic acid, the process is

referred to as anaerobic glycolysis. Lactate is
produced by erythrocytes, which lack mitochondria, and
by vigorously exercising muscle (where
oxygendemand>oxygensupply).
 In both cases, lactate is transported in the blood to the
liver, where it can provide substrate for
gluconeogenesis. The newly synthesized glucose
molecules can then be secreted by the liver into the
blood, where they can be taken up and oxidized by red
cells and muscle. This process is known as the Cori
cycle (Figure 6).

Lecture Title: Glycolysis and the oxidation of pyruvate 7

Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA
 Cancer cachexia

much used in the liver for gluconeogenesis

increase oxidation of metabolic fuels to provide

the ATP and GTP needed

increased oxygen consumption

Oxygen debt

 Lactate may be formed in the cytosol, but then enter the

mitochondrion to be oxidized to pyruvate for onward
metabolism – providing a pathway for transfer of
reducing equivalents from the cytosol into the
mitochondrion
Figure 7: Insulin promotes the translocation of GLUT 4 transporters from VI. THREE STEPS INVOLVING NONEQUILIBRIUM
intracellular vesicles in the plasma membrane of adipocytes and muscle REACTIONS
cells.
 Three enzymes are markedly exergonic and irreversible
reactions that are the major sites of regulation of
V. TISSUES THAT FUNCTION UNDER HYPOXIC
glycolysis (Figure 8):
CONDITIONS PRODUCE LACTATE 1. hexokinase (and glucokinase)
 True of skeletal muscles white fibers: 2. phosphofructokinase
o Rate of work output, and need for ATP formation 3. pyruvate kinase
may exceed the rate at which oxygen can be
taken up and utilized

 Tissues that normally derive much of their energy from

anaerobic glycolysis (produce lactate):
o Brain
o GI tract
o Renal medulla
o Retina
o Skin

 Tissues that take up and oxidize lactate but can produce

it under hypoxic conditions:
o Liver
o Kidneys
o Heart

 During high lactate production, as in:

o Vigorous exercise
o Septic shock

REVIEW!
How many ATP is produced directly by glycolysis? ___________
How many ATP is produced when NADH is included? ________
What glucose transporter is insulin dependent? _____________
What are the three cofactors of pyruvate kinase? ____________
TRUE or FALSE. Not all of the individual steps of glycolysis is
exergonic. ________

Figure 8: Regulation of hepatic glycolysis by metabolites.

Lecture Title: Glycolysis and the oxidation of pyruvate 8

Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA
 Phosphofructokinase
 Phosphofructokinase-1 (PFK-1) is the MAJOR
regulatory enzyme in glycolysis.
 Allosteric regulation of PFK-1 permits the enzyme to
respond to the energy needs of the cell and to hormonal
signaling by insulin and glucagon.
 PFK-1 is inhibited by:
o ATP
o Citrate, an intermediate of the TCA cycle
o glucagon
 PFK-1 is upregulated by:
o AMP or ADP
o Fructose 2,6-bisphosphate (F2,6P2 = F2,6-BP)
o Insulin
Fructose 2,6-Bisphosphate (F2,6P2)
 Allosteric activator of PFK-1
 Correlated with the insulin / glucagon ratio
 Two enzymes that determine the levels of F2,6P2 level
directly are phosphofructokinase-2 (PFK-2) and 2,6-
bisphosphatase (FBPase-2).
o The enzymes are regulated in such a way that
when one is active, the other is inactive. They
are encoded for by one gene.
o This is called a double-headed enzyme.
Figure 9: Hormonal regulation of the synthesis and catabolism of fructose
2,6-bisphosphate (F2,6P2) in liver. PFK-2, phosphofructokinase-2; FBPase-2,
fructose 2-6-bisphosphatase.
 The hepatic double-headed enzyme is regulated by
protein phosphorylation as shown above.
o When the protein is phosphorylated by cAMP-
activated protein kinase (cAMP-K), PFK-2 is
inactive and FBPase-2 is active, causing the
F2,6P2 level in the cell to decline.
o Glucagon (phosphorylates the double headed
enzyme), which activates adenylyl cyclase and
thus raises cAMP levels of the cell, therefore has
the effect of decreasing F2,6P2 level, decrease
PFK-1 activity, and ultimately slowing the flux of
glucose through glycolysis.
o Insulin, on the other hand, results in the activation
of phosphoprotein phosphatase that
dephosphorylates the double-headed enzyme,
Lecture Title: Glycolysis and the oxidation of pyruvate
Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA
thereby causing F2,6P2 concentration to rise and
increasing the flux of glucose through
glycolysis.
o In the liver, the protein constitutively synthesizes
F2,6P2 and glycolysis is not inhibited by
epinephrine-induced signaling.
 The reciprocal regulation of phosphofructokinase in
glycolysis and fructose 1,6-bisphosphatase in
gluconeogenesis is acted first on fructose that enters
glycolysis by phosphorylation to fructose 1-phosphate,
and bypasses the main regulatory steps, resulting in the
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.
Hexokinase
 Hexokinase is regulated by direct feedback inhibition
by one of the products of the reaction it catalyzes:
glucose-6-phosphate.
Pyruvate Kinase
 Catalyzes the last step of glycolysis
 Fructose 1,6-bisphosphate activation of pyruvate
kinase is an example of feed forward activation.
 Liver isozyme of pyruvate kinase is regulated by
phosphorylation and dephosphorylation (as discussed
above).
 Glucagon stimulates cAMP synthesis in hepatocytes,
causing cAMPK-A to phosphorylate pyruvate kinase.
 The phosphorylated form of protein kinase is inactive.
VII. THE OXIDATION OF PYRUVATE TO ACETYL-CoA
IS THE IRREVERSIBLE ROUTE FROM
GLYCOLYSIS TO THE CITRIC ACID CYCLE
What is the PDH Complex?
 The pyruvate dehydrogenase (PDH) complex and
the tricarboxylic acid (TCA) cycle account for the
complete combustion of pyruvate to CO2 and H2O.
 The main function of the PDH complex is to produce
acetyl-CoA, which can be oxidized completely to CO2
and water in the TCA cycle, and to generate NADH.
 The overall reaction of the PDH complex is:
pyruvate + NAD+ + CoASH
↓
Acetyl-CoA + NADH + H+ + CO2
 Conceptually, PDH is the bridge between glycolysis and
the TCA cycle. However, this bridge is unidirectional
because while humans can oxidize pyruvate to acetyl-
CoA (and ultimately to CO2), they cannot carry out the
opposite reaction of converting acetyl-CoA into
pyruvate.
o It is the irreversibility of the PDH complex that
explains why the liver cannot use acetyl-CoA as
a substrate for gluconeogenesis.
▪ Recall that acetyl-CoA is the product of fatty
acid β-oxidation
 Pyruvate is an important site of regulation. Inhibition of
PDH preserves glucose and gluconeogenic precursors
9
 when other fuels, such as acetyl-CoA generated by
oxidation of fatty acids, are available for utilization.

Pero ang akala ko sabi mo…?

These fatty acids are broken down to acetyl-CoA and undergo
the TCA cycle and ETC… not gluconeogenesis!

 Location: Associated with the matrix-facing surface

of the inner mitochondrial membrane.

Reactions of the PDH Complex

 Multienzyme subunit complex comprised of multiple

copies of each of three catalytic enzymes:
o Pyruvate dehydrogenase
o Dihydrolipoamide acetyltransferase
o Dihydrolipoamide dehydrogenase
 And two regulatory enzymes:
o PDH kinase
o PDH phosphatase
 Additionally it requires five cofactors:
o Coenzyme A (CoASH)
o NAD+
o FAD
o Lipoic acid
o Thiamine pyrophosphate

Steps involved in the PDH Complex (from Harper)

Figure 10: Oxidative decarboxylation by the pyruvate dehydrogenase
1. Pyruvate, formed in the cytosol, is transported complex. Lipoic acid is joined by an amide link to a lysine residue of the
into the mitochondrion by a proton symporter transacetylase component of the enzyme complex. It forms a long flexible arm,
2. Inside the mitochondrion, it is oxidatively allowing the lipoic acid prosthetic group to rotate sequentially between the active
decarboxylated to acetyl-CoA by a multienzyme sites of each of the enzymes of the complex.
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)
3. Pyruvate is decarboxylated by the pyruvate
dehydrogenase component of the enzyme
complex to a hydroxyethyl derivative of the
thiazole ring of enzyme-bound thiamin
diphosphate, which in turn reacts with oxidized
lipoamide, the prosthetic group of dihydrolipoyl
transacetylase, to form acetyl lipoamide.
4. Acetyl lipoamide reacts with coenzyme A to form
acetyl-CoA and reduced lipoamide. The reaction
is completed when the reduced lipoamide is
reoxidized by a flavoprotein, dihydrolipoyl
Figure 11: Sequence of reactions catalyzed by the PDH complex. TPP,
dehydrogenase, containing FAD. thiamine pyrophosphate; E1, pyruvate dehydrogenase subunit; E2,
5. Finally, the reduced flavoprotein is oxidized by dihydrolipoamide acetyltransferase subunit; E3, dihydrolipamide dehydrogenase.
NAD+, which in turn transfers reducing equivalents
to the respiratory chain. Thiamine

Review! • vitamin B1
• in deficiency: glucose metabolism is impaired, and there
What are the three enzymes that regulate glycolysis? What is the
MAIN rate limiting enzyme (encircle it)? is significant (and potentially life-threatening) lactic and
pyruvic acidosis
___________________________________________________
F2,6P2 is activated when the double headed enzyme is _________  The classical presentation of thiamine deficiency is
and is inhibited when it is ____________. (choices: phosphorylated beriberi.
| dephosphorylated)  A lack of dietary thiamine results in low levels of TPP
True or false, you can generate glucose from acetyl-CoA. ______ and impaired activity of the PDH, the α-ketoglutarate
dehydrogenase, as well as TPP-dependent
Lecture Title: Glycolysis and the oxidation of pyruvate 10
Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA
 transketolase, which is a component of the pentose
phosphate pathway.
 Thiamine deficiency is especially damaging to the heart
and brain, which have large energy requirements.
Pyruvate Dehydrogenase
• Regulated by End-Product Inhibition & Covalent
Modification
• inhibited by its products, acetylCoA, and NADH
• decreased activity: by phosphorylation (catalyzed by a
kinase) of three serine residues on the pyruvate
dehydrogenase component of the multienzyme
complex
• increased activity: and by dephosphorylation
(catalyzed by a phosphatase)
 The kinase is activated by increases in the [ATP]/
[ADP], [acetyl-CoA]/[CoA], and [NADH]/[NAD+] ratios.
Thus, pyruvate dehydrogenase, and therefore
glycolysis, is inhibited both when there is adequate
ATP available and also when fatty acids are being
oxidized.
• alcohol
- inhibits thiamine absorption, and may develop
potentially fatal pyruvic and lactic acidosis
Patients with inherited pyruvate dehydrogenase deficiency and
alcoholic patients have neurologic disturbances because of
the dependence of the brain on glucose as a fuel
Hemolytic anemia
• caused by inherited aldolase A deficiency and pyruvate
kinase deficiency in erythrocytes
 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, for example, during starvation,
when blood free fatty acid and ketone bodies are
increased, work capacity is improved.
REVIEW!
What are the three catalytic enzymes of the PDH complex?
________________________________________________
What are the two regulatory enzymes of the PDH complex?
________________________________________________
What are the five cofactors of the PDH complex?
________________________________________________
What is the main rate-limiting enzyme of the PDH complex? What
activates it? What inhibits it?
________________________________________________
________________________________________________
________________________________________________
What vitamin is important in the PDH complex?
________________________________________________

Figure 12: Regulation of PDH. Arrows with wavy shafts indicate allosteric
effects. (A) regulation by end product inhibition. (B) regulation by
intercovenrsion of active and inactive forms.

In fasting, when non-esterified fatty acid concentrations increase,

there is a decrease in the proportion of the enzyme in the active
form, leading to a sparing of carbohydrate. In adipose tissue, where
glucose provides acetyl-CoA for lipogenesis, the enzyme is
activated in response to insulin.

VIII. INHIBITION OF PYRUVATE METABOLISM LEADS

TO LACTIC ACIDOSIS
• pyruvate dehydrogenase is inhibited by arsenite and
mercuric ions that react with the ´SH groups of lipoic
acid and, as does a dietary deficiency of thiamin
(accumulation)

Lecture Title: Glycolysis and the oxidation of pyruvate 11

Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA
Lecture Title: Glycolysis and the oxidation of pyruvate 12
Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA
Lecture Title: Glycolysis and the oxidation of pyruvate 13
Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA
 QUIZ 8. The in-vitro conversion of glucose to glucose-6-
phosphate (glucose + Pi ↔ G6P + H2O) is highly
1. How many total molecules of ATP are synthesized
endergonic, through in the setting of glycolysis
from ADP via glycolysis of a single molecule of
proceeds in a spontaneous and irreversible fashion.
glucose?
How is this achieved?
a. 2
a. Endergonic reactions are always
b. 4
spontaneous and irreversible
c. 36
b. High concentrations of H2O shift equilibrium
d. 38
to favor G6P formation
2. Which of the following carbohydrates are the reactants
c. Through coupled ADP condensation
in glycolysis?
reactions
a. Glucose
d. Through coupled ATP condensation
b. Sucrose
reactions
c. A and B
9. Which of the following steps represent substrate-level
d. Neither A nor B
phosphorylation? The steps catalyzed by _________.
3. In which organism does glycolysis occur
I. – pyruvate kinase
a. Aerobic organisms
II. – G6PD
b. Anaerobic organisms
III. – PFK-1
c. A and B
IV. – phosphoglycerate kinase
d. Neither A nor B
a. I and IV only
4. Which statement concerning glycolysis and
b. I, II and III only
gluconeogenesis are is correct?
c. I only
a. Gluconeogenesis is catabolic and glycolysis
d. IV only
is catabolic
10. Which of the following enzymes represents the “lysis”
b. Gluconeogenesis is anabolic and glycolysis
portion of glycolysis?
is catabolic
a. PFK-1
c. Gluconeogenesis is catabolic and glycolysis
b. G6PD
is anabolic
c. Enolase
d. Gluconeogenesis is anabolic and glycolysis
d. Aldolase
is anabolic
11. Which of the following enzymes is BOTH a rate-
5. What body conditions favors gluconeogenesis over
limiting enzyme and catalyzes a substrate-level
glycolysis?
phosphorylation reaction?
a. High blood sugar
a. PFK-1
b. Starvation
b. Enolase
c. Low cellular levels of pyruvate
c. Pyruvate kinase
d. Increased cellular levels of AMP
d. Phosphoglycerate mutase
6. In the redox reaction pyruvate + NADH + H+ ↔ lactate
12. Which of the following is the MAIN key rate limiting
+ NAD+, which reactant is oxidized, and which is
enzyme of glycolysis?
reduced.
a. PFK-1
a. NADH is reduced and pyruvate is oxidized
b. Enolase
b. Lactate is reduced and NAD+ is oxidized
c. Pyruvate kinase
c. Lactate is reduced and pyruvate is oxidized
d. Phosphoglycerate mutase
d. Pyruvate is reduced and NADH is oxidized
13. Which of the following is NOT a cofactor of the
7. What is the best description for the energetics of
conversion of PEP to pyruvate?
glycolysis?
a. Mg2+
a. Overall under typical cellular conditions,
b. Mn2+
glycolysis has a negative free-energy
c. K+
change, though there are steps of both
d. Vitamin B1
positive and negative free-energy change.
14. Which of the following does not belong to the group?
b. Overall under typical cellular conditions,
glycolysis has a positive free-energy a. Pyruvate dehydrogenase
change, though there are steps of both b. Pyruvate dehydrogenase kinase
positive and negative free-energy change. c. Dihydrolipoamide acetyltransferase
c. ALL the steps of glycolysis have a negative d. Dihydrolipoamide dehydrogenase
free-energy change 15. Where does metabolism of pyruvate to acetyl-CoA
d. ALL the steps of glycolysis have a positive occur?
free-energy change a. In matrix-facing surface of the inner
mitochondrial membrane
b. In the mitochondrial matrix
c. In the cytosol
d. In the inner mitochondrial membrane

BACBBDADADCADBA

Answers:

Lecture Title: Glycolysis and the oxidation of pyruvate 14

Module: 3.1.1
Transcribed by: LEONCIO, PAMITTAN, YAO | Checked by: MOLINA & URSUA