Module 2

Glycolysis and the Catabolism of Hexoses

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Fermentation in cell-free yeast
extracts (1897)
Glycolysis
Sugar as metabolic fuels
Pentose phosphate pathway
Overview of Glycolysis
Glycolysis (lysis of glucose)
Takes place in the cytoplasmof eukaryotic cells.
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Glycolytic enzymes
located in the cytosol.
loosely associated (if at all) with
cell structures such as membranes.
Overview of Glycolysis
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Glycolysis is anaerobic.
Theory: glycolysis evolved before substantial
amounts of oxygen accumulated in the
atmosphere.
Primitive Earth
Most living things metabolize glucose by identical pathways.
They share a common biochemistry in spite of their enormous
diversity.
Overview of Glycolysis
Glycolysis (lysis of glucose)
Breakdown of a glucose (6C) into two molecules of
pyruvate (3C).
Some free energy released from glucose is conserved in
the form of ATP and NADH.
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Glucose Pyruvate
Chemical strategy of glycolysis
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Add phosphoryl
groups to the
glucose.
Chemically
convert
phosphorylated
intermediates into
compounds with
high phosphate
group-transfer
potentials.
Chemically couple
the subsequent
hydrolysis of
reactive
substances to ATP
synthesis.
The pathway is composed of chemically coupled
phosphoryl-transfer reactions.
Importance of phosphorylated intermediates
Conservation of metabolic energy
energy from ATP is partially conserved in high energy
phosphate compounds.
e.g. 1,3-bisphophoglycerate, phosphoenolpyruvate.
Less energy is consumed
to retain phosphorylated intermediates in the cell.
plasma membrane generally lacks transporters for
phosphorylated sugars.
Binding energy
between phosphate groups and active sites of enzymes.
lowers the activation energy and increases the
specificity of the enzymatic reactions.
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Glycolysis has two phases
Preparatory phase (5 steps)
The hexose chain is cleaved into two triose
phosphates.
Two molecules of ATP are invested.
Payoff phase (5 steps)
Conversion of two molecules of glyceraldehyde 3-
phosphate (G3P) to two molecules of pyruvate.
Formation of four molecules of ATP and two
molecules of NADH.
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Net yield of ATP: two ATP per molecule of glucose converted to pyruvate.
The overall balance sheet show a net gain of ATP
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Glucose + 2ATP + 2NAD
+
+ 4 ADP + 2P
i

2 pyruvate + 2ADP + 2NADH + 2H
+
+4ATP + 2H
2
O
Glucose + 2NAD
+
+ 2ADP + 2P
i

2 pyruvate + 2NADH + 2H
+
+ 2ATP +2H
2
O
Note: Glycolysis is an essentially irreversible
process driven to completion by a large net
decrease in free energy.
G = -85 kJ/mol
The oxidizing agent of glycolysis
NAD
+
: the primary oxidizing agent of glycolysis.
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Glucose + 2NAD
+
+ 2ADP + 2P
i

2 pyruvate + 2NADH + 2H
+
+ 2ATP +2H
2
O
Note: The NADH produced must be
continually reoxidized to keep the
pathway supplied with NAD
+
.
NADH NAD
+
Oxidation
Reduction
Recycling of NAD
+
Aerobic conditions
Mitochondrial oxidation of each NADH to
NAD
+
yields 2.5ATPs.
Anaerobic conditions
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Muscle(homolactic fermentation)
NAD
+
is regenerated when NADH
reduces pyruvate to lactate.
Yeast (alcoholic fermentation)
Pyruvate is decarboxylated to yield CO
2
and acetaldehyde.
Acetaldehyde is reduced by NADH to
yield NAD
+
and ethanol.
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Possible catabolic
fates of the pyruvate
formed in glycolysis
As precursor in
anabolic reactions
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1. Phosphorylation of glucose
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Glucose is activated for subsequent reactions by its phosphorylation
at C-6 to yield glucose 6-phosphate, with ATP as the phosphoryl donor.
This reaction is irreversible under intracellular conditions.
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The nucleophilic attack of the C6-OH group of glucose on the
phosphate of an Mg
2+
-ATP complex.
Mg
2+
shields the negatively charged groups of ATP and thereby
facilitate the nucleophilic attack.
Mechanism of the hexokinase reaction
Hexokinase and glucokinase
Hexokinase
A relatively nonspecific enzyme contained in all cells.
Substrates
Hexoses (e.g D-glucose, D-mannose, D-fructose).
Mg
2+
-ATP complex.
Uncomplexed ATP is a potent competitive inhibitor of hexokinase.
Glucokinase
Liver cells also contain glucokinase.
Catalyzes the same reaction, but primarily involved
in the maintenance of blood glucose levels.
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2. Conversion of glucose 6-phosphate to
fructose 6-phosphate
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Reversible isomerization.
C-1 : carbonyl alcohol (prepare for phosophorylation at C-1)
C-2 : alcohol carbonyl (prepare for cleavage of bond between C-3 and C-4)
(Aldose) (Ketose)
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The phosphohexose isomerase reaction
3. Conversion of Fructose 6-phosphate
to fructose 1,6-bisphosphate
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The second ATP utilization (Transfer of a phosphoryl group from ATP
to F6P.)
Essentially irreversible.
The first committed step in the glycolytic pathway.
(PFK-2 catalyzes the formation of Fructose2,6-bisphosphate)
Importance of PFK-1
PFK plays a central role in the control of glycolysis.
It catalyzes one of the pathways rate-determining
reactions.
Activity of PFK is subject to complex allosteric
regulation.
enhanced allosterically by several substances, including
AMP;
inhibited allosterically by several other substances,
including ATP and citrate.
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4. Cleavage of fructose 1,6-bisphosphate
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(aldose) (ketose)
A lower concentration of reactants present in cells, the
actual free-energy is small and the aldolase reaction is
readily reversible.
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The end product of the two reactions is glyceraldehyde 3-
phosphate (two molecules).
Fate of the glucose carbons in
the formation of glyceraldehyde
3-phosphate
5. Interconversion of the triose phosphate
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Dihydroxyacetone phosphate is rapidly and reversibly converted to
glyceraldehyde 3-phosphate.
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The carbon atoms derived from C-1, C-2 and C-3 of the starting glucose
are chemically indistinguishable from C-6, C-5, and C4, respectively.
Each carbon of glyceraldehyde 3-phosphate is derived
from either of two specific carbons of glucose.
6. Oxidation of glyceraldehyde 3-
phosphate to 1,3-bisphosphoglycerate
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The first of the two energy-conserving reactions of
glycolysis that eventually lead to the formation of ATP.
Much of the energy is conserved by the formation of the acyl
phosphate group (carboxylic acid anhydride with phosphoric acid)
at C-1 of 1,3-bisphophoglycerate.
Significance of acyl phosphate formation
Aldehyde oxidation, an exergonic reaction, drives
the synthesis of acyl phosphate.
Acyl phosphate has a very high standard free
energy of hydrolysis. (G = -49.3 kJ/mol)
For the formation of ATP.
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1,3-Bisphosphoglycerate
Note:
Amount of NAD
+
in a cell (10
-5
M).
The NADH formed is continuously reoxidized
and recycled (else glycolysis will soon come
to a halt).
7. Phosphoryl transfer from 1,3-
bisphosphglycerate to ADP
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Formation of the first ATP.
The enzyme phosphoglycerate
kinase transfers the high-energy
phosphoryl group from the carboxyl
group of 1,3-bisphoglycerate to ADP,
forming ATP and 3-
phosphoglycerate.
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Mechanism of the phosphoglycerate kinase (PGK) reaction
8. Conversion of 3-phosphoglycerate to
2-phosphoglycerate
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The enzyme phosphoglycerate mutase catalyzes a reversible shift of the
phosphoryl group between C-2 and C-3 of glycerate.
This reaction is necessary preparation for the next reaction which
generates a high-energy phosphoryl compound for use in ATP synthesis.
9. Dehydration of 2-phosphoglycerate
to phosphoenolpyruvate
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The second high-energy intermediate formation.
Enolase promotes reversible removal of a molecule of water from 2-
phosphoglycerate to yield phosphoenolpyruvate (PEP).
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A compound with a relatively low phosphoryl group transfer
potential is converted to one with high phosphoryl group transfer
potential.
G for hydrolysis of 2-
phosphoglycerate is -17.6kJ/mol.
G for PEP hydrolysis is -61.9kJ/mol.
Insufficient to t drive ATP synthesis (G= 30.5 kJ/mol)
10. Transfer of the phosphoryl group
from phosphoenolpyruvate to ADP
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The last step in glycolysis.
Spontaneous conversion of the
enol form of pyruvate to the keto
form.
a large negative standard free
energy change in the overall
reaction.
PEP hydrolysis (G=-61.9 kJ/mol)
Used in ATP formation (G= -30.5 kJ/mol)
Used in driving force (G= -31.4 kJ/mol)
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The product pyruvate first appears in its enol form, then tautomerizes
rapidly and nonenzymatically to its keto form which predominates at
pH7.
Glycolytic pathway is tightly controlled
The adjustment in the rate of glycolysis is
achieved by a complex interplay among different
factors:
ATP consumption
NADH regeneration
Allosteric regulation of several glycolytic enzymes:
Hexokinase, PFK-1, and pyruvate kinase.
Fluctuations of metabolites
Hormones
Glucagon, epinephrine, and insulin.
Expression of the genes for several glycolytic enzymes.
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Glycolysis in Muscle
Glycolysis in muscle is regulated to meet the
need for ATP.
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The ATP is primarily provided to
power muscle contraction.
Glycolysis in muscle is regulated to
meet the need for ATP
The primary control of muscle glycolysis is the energy
charge of the cell (the ratio of ATP to AMP).
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The energy charge can have a value ranging from 0 (all AMP)
to 1 (all ATP).
Note: Index of the energy status in the energy charge, which is proportional
to the mole fraction of ATP plus half the mole fraction of ADP, given that
ATP contains two anhydride bonds, whereas ADP contains one.
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Energy charge regulates metabolism
High [ATP] inhibits the relatives rates of a typical ATP-generating
(catabolic) pathway and stimulate the typical ATP-utilizing
anabolic pathway.
The energy charge (like the
pH of a cell), is buffered.
The pathway is controlled
to maintain the energy
charge within rather narrow
limits (0.80-0.95).
The curves are steep near an
energy charge of 0.9, where
they usually intersect.
Phosphofructokinase
Phosphofructokinase is the most important control
site in the mammalian glycolytic pathway (First
committed step).
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Role of ATP in PFK-1 reaction:
i. Substrate
ii. End product
Structure of phosphofructokinase
Tetramer of four identical subunits
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High [ATP]: when ATP production > consumption
ATP inhibits PFK-1 by binding to an allosteric site and lowering
the affinity of the enzyme for its substrate fructose 6-phosphate.
Result:
A high [ATP] converts the hyperbolic
binding curve of F6P into a sigmoidal
one.
Protection of muscle: A decrease in pH also inhibits PFK activity by augmenting the
inhibitory effect of ATP. (The pH might fall when muscle is functioning anaerobically,
producing excessive quantities of lactic acid.)
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At rest, glycolysis is not
very active (thin arrows).
The high [ATP] inhibits
PFK (also pyruvate
kinase & hexokinase).
Note: Glucose 6-phosate
is converted into glycogen.
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Low [ATP]: when ATP consumption > production
AMP (also ADP) increases. AMP acts allosterically to relieve this
inhibition by ATP.
Result:
The activity of the enzyme increases
when the ATP/AMP ratio is lowered.
i.e. Glycolysis is stimulated as the
energy charge falls.
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During exercise, the
decrease in the
ATP/AMP ratio
resulting from muscle
contraction activates
phosphofructokinase
and hence glycolysis.
Note: The flux down the
pathway is increased, (thick
arrows).
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Some regulators affecting PFK-1 activity
Note: AMP is a better positive regulator of PFK than ADP.
When ATP is being utilized rapidly, adenylate kinase converts ADP to ATP.
AMP becomes the signal for
the low-energy state.
ADP + ADP ATP + AMP
In a typical cell, [ATP] > [ADP] > [AMP]. Small % change of ATP causes
large % change of AMP (i.e. control of PFK is sensitive).
Regulation of pyruvate kinase and hexokinase
Pyruvate kinase is
allosterically inhibited by
ATP.
to slow glycolysis when
the energy charge is high.
Hexokinse is inhibited by
its product (G6P).
High [G6P] signal that the
cell no longer requires for
energy or for the synthesis
of glycogen.
Note: G6P can also be
converted to glycogen in
muscle.
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The Liver has diverse biochemical functions.
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The regulation of glycolysis in the liver
The regulation of glycolysis in liver
is more complex than muscle.
It plays a major role in:
Metabolism
Glycogen storage
Detoxification
Decomposition of red blood cells
Plasma protein synthesis
Hormone production, etc.
Three major signals for the regulation
of glycolysis in liver
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Glycolysis
in liver
Availability of
biosynthetic
precursor
Energy need
(ATP)
Note: Regulation
with respect to ATP
is the same in the
liver as in muscle.
Note: Furnish carbon
skeletons for biosyntheses.
Responses to changes in blood
glucose (maintain blood glucose level)
The regulation of glycolysis in the liver
Phosphofructokinase (PFK)
i. PFK is inhibited by citrate.
Citrate
an early intermediate in the citric acid cycle.
a key intermediate in the aerobic oxidation of pyruvate,
fatty acids, and amino acids.
A high level of citrate in the cytoplasm
biosynthetic precursors are abundant.
no need to degrade additional glucose for this purpose.
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Note: Citrate inhibits PFK by enhancing the inhibitory effect of ATP,
further reducing the flow of glucose through glycolysis.
The regulation of glycolysis in the liver
Phosphofructokinase (PFK)
ii. PFK is activated by F-2,6-BP.
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Note: In high concentrations, fructose 6-phosphate (F-6P)
activates the PFK through an intermediary, F-2,6-BP.
Actions of F-2,6-BP
Increases the affinity of PFK for F6P;
Diminishes the inhibitory effect of ATP.
PFK is activated by fructose 2,6-bisphosphate (F-2,6-BP)
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Sigmoidal dependency
becomes hyperbolic.
Allosteric inhibitory effect of high
[ATP] is reversed by F-2,6-BP.
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Feed-forward stimulation
Glycolysis is accelerated
when glucose is abundant
[Glucose]
[F-6P]
[F-2,6-BP]
PFK activity
Glycolysis
Result: [Glucose]
Response to the
change of blood
glucose level
PFK is activated by fructose 2,6-bisphosphate (F-2,6-BP)
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Summary of the regulators affecting PFK-1 activity
Glucokinase: isozyme of hexokinase in liver
Glucokinase
Monitor of blood glucose levels.
Provide glucose 6-phosphate (G6P) for the synthesis of
glycogen and fatty acids.
Features of glucokinase
Not inhibited by G6P.
Affinity for glucose is 50-fold lower than that of
hexokinase.
Phosphorylates glucose only when [glucose] is high.
(i.e. glucose will not be wasted when it is abundant)
Brain and muscle can obtain glucose when its supply is limited.
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Note: The hexokinase reaction in the liver is controlled as in the muscle.
Pyruvate kinase
Isozymes of pyruvate kinase.
L type: predominates in liver.
M type: predominates in muscle and brain.
Both isozymic forms are subject to the
allosteric regulation similarly.
The L type (but not M type) is also controlled
by reversible phosphorylation.
Prevents liver from consuming glucose when it is
more urgently needed by the brain and muscle.
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Control of catalytic activity of pyruvate kinase
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Note: Pyruvate kinase is regulated by allosteric effectors and
covalent modification.
Sugar as metabolic fuels
Other carbohydrates (besides glucose)
Glycogen & starch.
Maltose, lactose, sucrose.
Fructose, mannose, galactose, etc.
Feeder pathways for glycolysis
Different carbohydrates are transformed into one
of the glycolytic intermediates.
Integrate into the catabolic pathway of glycolysis.
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Entry of different carbohydrates
into the preparatory stage of
glycolysis
Pentose Phosphate Pathway (PPP) of
Glucose Oxidation
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Oxidative pathway
NADP
+
is the electron acceptor.
NADPH is yielded.
NADPH
Ribose 5-phosphate
CO
2
Production of specialized
products needed by the cell.
PPP
In rapidly dividing cells (e.g. bone marrow,
intestinal mucosa, and cancer), the ribose 5-
phosphate is used to make RNA and DNA, etc.
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General scheme of
the PPP
Nonoxidative phase
occurs in cells that
are not using ribose
5-phophate for
biosynthesis.
Importance of NADPH as an electron donor
NADPH is required for reductive biosynthesis
Tissues that carry out extensive fatty acid synthesis.
e.g. liver, adipose, lactating mammary gland.
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Importance of NADPH as an electron donor
NADPH is required for reductive biosynthesis
Tissue that carry out very active synthesis of
cholesterol and steroid hormones.
e.g. liver, adrenal glands, gonads.
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NADPH is required to counter the damaging
effects of oxygen radicals.
Erythrocytes and the cells of the lens and cornea are
directly exposed to oxygen and thus to the damaging free
radicals generated by oxygen.
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Importance of NADPH as an electron donor
Note: A reducing cellular condition can prevent or undo oxidative
damage of proteins, lipids, and other sensitive molecules in cells.
A reducing condition is needed
high ratio of NADPH/ NADP
+
high ratio of reduced to oxidized
glutathione.
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Oxidative reactions of the PPP
End products: Ribose 5-phosphate, CO
2
,
and NADPH.
Note: All the enzymes in the PPP are located
in the cytosol.
Fates of Glucose 6-phosphate
Two major factors:
current needs of the cell.
[NADP
+
] in the cytosol.
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Example:
NADPH is rapidly converted to NADP
+
in biosynthetic reductions.
[NADP
+
] rises.
G6PD is stimulated allosterically.
The flux of G6P through the PPP is increased.
G6P
Glycolysis
Pentose phosphate pathway
?
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Example:
NADPH is forming faster than it
is being used for biosynthesis
and glutathione reduction.
[NADPH] rises and inhibits the
first enzyme in the PPP.
more G6P is available for
glycolysis.
Partitioning of G6P between glycolysis and the PPP
Role of NADPH
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- End of Module 2