Glycolysis

5/9/03

Pathway overview
1. Add phosphoryl groups to activate glucose.
2. Convert the phosphorylated intermediates into high energy
phosphate compounds.
3. Couple the transfer of the phosphate to ADP to form ATP.
Stage I A preparatory stage in which glucose is phosphorylated
and cleaved to yield two molecules of glyceraldehyde-3phosphate - uses two ATPs
Stage II glyceraldehyde-3-phosphate is converted to pyruvate
with the concomitant generation of four ATPs-net profit is
2ATPs per glucose.

Glucose + 2NAD+ + 2ADP +2Pi 2NADH +

2pyruvate + 2ATP + 2H2O + 4H+

Front half of glycolysis

Phosphoglycerate Kinase: First ATP

generation step
OPO32-

O
H

OH + ADP

CH2OPO32-

1,3 BPG

O-

O
H

OH + ATP

CH2OPO323 PG

GAP + Pi + NAD+

1,3 -BPG + NADH + 6.7 kJ mol-1

1,3 BPG + ADP

3PG + ATP

-18.8 kJ mol-1

GAP+Pi+NAD+ +ADP 3PG+NADH+ATP -12.1 kJmol-1

Phosphoglycerate mutase
O-

O
H

O-

OH

CH2OPO32-

O-

CH2OH

2PG

3 PG
H

OPO3-2

CH2OPO32-

2,3 BPG

OPO3-2

Phosphoglycerate mutase requires a phosphorylated

form of the enzyme to be active. Only 2,3 BPG can
phosphorylate the unphosphorylated enzyme.
N
Enzyme

H2 C

N
PO32-

Phospho Histidine residue

Glycolysis influences oxygen transport

Oxygen saturation curves in erythrocytes

Enolase generation of a second high

energy intermediate
O-

O
H
H

OPO3-2

OH

2 Phosphoglycerate

O-

OPO3-2 + H2O

C
H

Phosphoenol pyruvate

Pyruvate kinase: Second ATP

generation step

The second half of glycolysis

The metabolic fate of pyruvate

The need to regenerate NAD+ from NADH

A. Homolactic fermentation: conversion of pyruvate
to lactate
O

O
C

HS

HR

O
NH2

CH3

LDH
HO

H+
CH3

Pyruvate

NADH

O-

O
NH2

+
N
R

L-Lactate

NAD+

Mammals have two different types of enzymes: Isozymes

M type for muscle
H type for heart

Lactate dehydrogenase is a tetramer H4 has a low

Km for pyruvate and is allosterically inhibited by
high concentrations of pyruvate.

M4 has a higher Km for pyruvate and is not

allosterically regulated
Although all five types can exist, H4, H3M, H2M2
HM3, M4 The M predominates in anaerobic muscle
tissues which favor the formation of lactate while the
H4 form predominates in aerobic tissues like heart
where the formation of pyruvate from lactate is
preferred

Pro-R hydride is
transferred from C4 of
NADH to C2 of pyruvate
with the concomitant
transfer of a proton from
His 195
All muscle lactate is transferred to
the liver where it is turned back to
glucose

Alcoholic fermentation

A two step process:

1) Pyruvate decarboxylase requires thiamine
pyrophosphate TPP as a cofactor.
2) Alcohol dehydrogenase requires Zn+2 as a cofactor

Thiamine pyrophosphate

The build up of negative charges seen in decarboxylation

reactions on the carbonyl atom in the transition state is
unstable and TPP helps stabilize the negative charge

Reaction mechanism of pyruvate

decarboxylation
1. Nucleophilic attack by
the ylid from of TPP on
the carbonyl
2. Departure of CO2 and
resonance-stabilization
of the carbanion.
3. Protonation of the
carbanion
4. Elimination of TPP
ylid to form
acetaldehyde

Long distance hydrogen

bonding and general acid
catalysis from Glu 51 with
the aminopyrimidine ring
leads to the formation of
the ylid form of TPP.

Deficiencies of TPP lead to Beriberi

Vitamin B1
Beriberi was prevalent in the rice consuming countries of
the Orient where polished rice is preferred. TPP is found
in the brown outer layers of rice.
Neurological atrophy, cardiac failure, endema nowadays
found in alcoholics who would rather drink than eat.

Alcohol dehydrogenase

Energetics of Fermentation
G'
Glucose

2lactate + 2H+

Glucose

2CO2 + 2ethanol -235 kJ mol-1 of glucose

Formation of 2ATP

-196 kJ mol-1 of glucose

+61 kJ mol-1 of glucose

equals 31% and 26% efficient for energy conservation

Under physiological conditions this efficiency
approaches 50%

Glycolysis is for rapid ATP production

Glycolysis is about 100 times faster than oxidativephosphorylation in the mitochondria
Fast twitch muscles - short blasts of energy and are
nearly devoid of mitochondria use exclusively glycolysis
for ATP
Slow twitch muscles are dark red, rich in mitos obtain
ATP from OX-phos., i.e. flight muscles of migratory
birds and the muscles of long distance runners

Control of Metabolic Flux

J

vj

S AB P
vr

J vf - v r

At equilibrium J = 0 and far from equilibrium J=vf

The flux throughout the pathway is constant at steady
state conditions and control of flux requires:
1) The flux-generating step varies with the organisms
metabolic needs
2). The change in flux is felt throughout the pathway

A diagrammatic representation of substrate cycling

and control of flux

An increase in flux, J causes [A] to increase and

when [A] increases the is a vf So J = vf

J v f v f v f
vf

J
vf J
vf vf v r
Plugging in the the Michaelis -Menton equation

V max A
V f max A
vf
if [A] K M
vf
KM
K M A
f

v f A
V f max A
v f
hence

KM
vf
A

J A
vf

J
A v f vr

vf/(vf-vr) is a measure of the sensitivity of a reactions

fractional change in flux to its fractional change in
substrate concentration
1. In an irreversible reaction, vr approaches 0 and vf/(vf-vr)
approaches 1. The reaction is therefore requires a nearly
fractional increase in its substrate concentration order to
respond to a fractional increase in flux
2. As a reaction approaches equilibrium, vr approaches vf and
vf/(vf-vr) approaches infinity. The reactions response to a
fractional increase in flux therefore requires a much smaller
fractional increase in its substrate concentrations.

Flux is controlled at the rate limiting step

Usually the product is removed much faster than it is
formed so that the rate-determining step is far from
equilibrium. Because of the fractional change in the
flux J/J when vf>>vr is directly proportional to the
change is substrate concentrations other mechanisms
are needed to achieve factors of over 100 as seen in
glycolysis.
Allosteric regulation
Covalent modification
Substrate cycling
Genetic control

Covalent modification-Protein phosphorylation

Three steps to elucidate common

controlling mechanisms in a pathway
1. Identify the rate determining steps: Those with a
large negative G and measure flux through the
pathway and each step with inhibitors.
2. Identify In vitro allosteric modifiers of the pathway
study each enzymes kinetics, mechanisms and
inhibition patterns.
3. Measure in vivo levels of modulators under
conditions consistent with a proposed control
mechanism

Free energy changes in glycolysis

Reaction

enzyme

Hexokinase

-20.9

-27.2

PGI

+2.2

-1.4

PFK

-17.2

-25.9

Aldolase

+22.8

-5.9

TIM

+7.9

+4.4

6+7 GAPDH+PGK
PGM

-16.7
+4.7

-1.1
-0.6

Enolase

-3.2

-2.4

10

PK

-23.3

-13.9

Only three enzymes function with large negative Gs

Hexokinase, Phosphofructokinase and pyruvate kinase
The other enzymes operate near equilibrium and their
rates are faster than the flux through the pathway.
Specific effectors of Glycolysis
Enzymes

Hexokinase
PFK

Inhibitors

Activators

G6P

none

ATP, citrate, PEP

ADP, AMP, cAMP

FBP,F2,6BP, F6P
NH4, Pi

PFK: the major flux controlling enzyme of

glycolysis in muscle

PFK activity as a function of G6P

AMP concentrations not ATP control

glycolysis
ATP concentrations only vary about 10% from resting to active
cells. [ATP] is buffered by creatine phosphate and adenylate
kinase.

2ADP

ATP + AMP

ATP AMP
K
0.44
2
ADP
A 10% decrease in ATP produces a four fold increase in AMP
because ATP = 50AMP in muscle. AMP activates PFK by the
action of adenylate kinase.

Substrate cycling
Fructose-6 phosphate +ATP

Fructose 1,6-bisphosphate

Fructose 1,6-bisphosphate

Fructose-6 phosphate + Pi

The net result is the breakdown of ATP.

Two different enzymes control this pathway PFK and
Fructose 1,6 bisphosphatase. If these both were not
controlled a futile cycle would occur.
Specific effectors of Glycolysis
Enzymes

Inhibitors

Activators

Phosphatase

AMP

ATP, citrate