BAHAN KULIAH BIOKIMIA POWER POINT BAGIAN 1 /BIOCHEMISTRY POWER POINT LECTURES PART 1 | Karya Tulis Ilmiah

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

C

OH + ADP

CH2OPO32-

1,3 BPG

O-

O
H

C

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 kJ•mol-1

Phosphoglycerate mutase
O-

O

H

C

O-

O

OH

H

CH2OPO32-

O-

O

C

CH2OH

2PG

3 PG
H

C

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

H2C

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

C

OPO3-2

C

OH

H

2 Phosphoglycerate

O-

O

OPO3-2 + H2O

C
H

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

-

O

HS

HR

O
NH2

+

CH3

N

+

LDH
HO

C

H

H+
CH3

R

Pyruvate

NADH

H

O-

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

J

S   A  B   P
vr

J v f - 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

f

V max  A 
vf 
K M   A

V f max A
vf 
KM

if [A]  K M

 
 

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 reaction’s 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

G´

G

1

Hexokinase

-20.9

-27.2

2

PGI

+2.2

-1.4

3

PFK

-17.2

-25.9

4

Aldolase

+22.8

-5.9

5

TIM

+7.9

+4.4

6+7 GAPDH+PGK
PGM

-16.7
+4.7

-1.1
-0.6

8

9

Enolase

-3.2

-2.4

10

PK

-23.3

-13.9

Only three enzymes function with large negative G’s
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