Download Regulation of Glycolysis

Regulation of Glycolysis
January 27, 2003
Bryant Miles
Because the principle function of glycolysis is to produce ATP, it must be regulated so that ATP is
generated only when needed. The enzyme which controls the flux of metabolites through the glycolytic
pathway is phosphofructokinase (PFK-1). PFK-1 is an allosteric enzyme that occupies the key
regulatory position for glycolysis. PFK-1 has a tetrameric enzyme composed of four identical subunits.
Like other allosteric proteins (hemoglobin) and enzymes (ATCase) the binding of allosteric effectors
and substrates is communicated to each of the active sites. Quaternary changes are concerted and
preserve the symmetry of the tetramer. PFK-1 has two sets of alternative interactions between subunits
which are stabilized by hydrogen bonds and electrostatic interactions. The two set of conformations are
called the T and R states.
These two conformational states are in equilibrium: T
R
I. ATP feedback inhibition. The function of the glycolytic pathway is to generate ATP. ATP is both a
substrate and an allosteric inactivator. The enzyme has two
binding sites for ATP. One is the substrate binding site and
the other one is an inhibitory site. The PFK-1 substrate
binding site binds ATP equally well in both the T and R
states. The inhibitory ATP binding site only binds ATP
when the enzyme is in the T conformation. The other
substrate fructose-6-phosphate binds only to the R state.
High concentrations of ATP shift the equilibrium towards
the T conformation which decreases the affinity of the
enzyme for F-6-P.
II. AMP reverses inhibition. AMP reverses the inhibition due to high concentrations of ATP. AMP
binds preferentially to the R state of PFK. This is important the concentration of ATP drops only 10 %
during vigorous exercising. AMP concentration levels in the cell can rise dramatically due to the
enzymatic activity of adenylate kinase.
2ADP
K 'eq =
ATP + AMP
[ ATP ][ AMP]
[ ADP] 2
The steady state concentration of ATP in the cell is 10 times greater than the concentration of ADP, and
50 times the concentration of AMP. As a result of the activity of adenylate kinase, a 10% decrease in
the concentration of ATP results in 400 % increase in the concentration of AMP.
III Other allosteric effectors of PFK-1
ADP is another allosteric effector of PFK-1. ADP like AMP reverses the inhibitory effects of ATP and
is therefore considered an allosteric activator. The activity of PFK-1 is dependent on the ATP, ADP and
AMP concentrations which are all functions of the cellular energy status.
HO
H2C
CO2-
C
CO2
H2C
CO2-
-
Under aerobic conditions, the pyruvate formed by glycolysis is fed into the citric
acid cycle where it is completely oxidized into CO2 and H2O. Citrate is a
metabolite of the citric acid cycle. When the citric acid cycle is saturated,
glycolysis needs to be slowed down. When the citric acid cycle is saturated, the
citrate concentration in the cytosol increases. Citrate binds preferentially to the Tstate of PFK-1. Thus high concentrations of citrate inactivate the enzyme.
Citrate
PFK-1 is also regulated by β-D-fructose 2-6-bisphosphate
which is a potent allosteric activator of the enzyme. β-Dfructose 2-6-bisphosphate binds to the R-state of the enzyme
and increases the affinity of the enzyme for fructose-6phosphate.
β-D-fructose 2-6-bisphosphate also decreases the inhibitory effects of ATP
OO
O P O CH
2
O
OH
OH
H
HO
H
-
O
P
O
H2C OH
β-D-Fructose-2,6-bisphosphate
O
Metabolism of Hexoses other than glucose.
I. Fructose.
Sucrose is a major fuel source of our diets. Sucrose is a dissacharide of fructose and glucose. There are
two pathways for the metabolism of fructose.
H O
OMg
O P O CH
H C OH
CH2
2 O H2C OH
O 2
Hexokinase OH
OH
H
OH
H
OH
H
O
ATP ADP
H
HO
H H
HO
ATP
2+
Mg2+
PFK-1
ADP
-
O
O P O
O- H
O
H
H
H
HO
O
H2C O P OOH
H
OH
O
-
The first and simplest pathway for fructose
catabolism occurs in the muscle cells where
fructose is a substrate for hexokinase which
produces fructose-6-phosphate, a substrate for PFK1.
The liver, however, contains little hexokinase. Instead it contains glucokinase which specifically
phosphorylates glucose. The liver catabolizes fructose through a pathway that involves six enzymes.
H O
H C OH
CH2
O 2
H
OH
H
HO
OH
H
ATP
Fructokinase
ADP
H O
O
CH2O P O-
O
H
HO
H
O-
C O
H C O P OCH2
O 2
OH
OH
HO C H
H C OH
OH
H C OH
CH2OH
Fructose-1-phosphate aldolase
ADP
O
CH2OH
CH
H C OH
CH
H C OH
ATP
Glyceraldehyde Kinase
O
H2C O P OO-
TIM
NAD+
NADH
O
Alcohol Dehydrogenase
CH2OH
CH2OH
+
H C OH
ATP
Glycerol
Kinase
O
ADP
CH2O P O
C O
- -
O
NADH
NAD
+
CH2OH
CH2OH
H C OH O
Glycerol Phosphate
Dehydrogenase
H 2C O
P OO-
Step 1:
In the liver the first step of fructose catabolism is the phosphorylation of fructose by fructokinase to
form fructose-1-phosphate. Note that neither hexokinase or phosphofructokinase can phosphorylate
fructose-1-phosphate into fructose-1,6-bisphosphate.
Step 2:
In the liver we find a class I aldolase which is an isozyme of fructose bisphosphate aldolase (aldolase
Type A). The Type A aldolase is specific for the substrate fructose-1,6-bisphosphate. The isozyme of
aldolase found in the liver is called a Type B aldolase. It can utilize fructose-1-phosphate as well as
fructose-1,6-bisphosphate as a substrate. So the Type B aldolase found in the liver catalyzes the
aldolytic cleavage of fructose-1-phosphate into glyceraldehydes and dihydroxyacetone phosphate.
Step 3:
The dihydroxyacetone phosphate produced can be converted into glyceraldehyde 3-phosphate by the
reaction catalyzed by triose phosphate isomerase. The other product of the aldolytic cleavage,
glyceraldehydes can be directly phosphorylated by glyceraldehyde kinase to form glyceraldehydes 3phosphate.
Step 4: There is an alternative pathway where the glyceraldehydes is reduced by alcohol dehydrogenase
into glycerol. Glycerol is then phosphorylated by glycerol kinase to form glycerol-3-phosphate. The
glycerol 3-phosphate is then oxidized by glycerol phosphate dehydrogenase into dihydroxyacetone
phosphate which of course is converted into glyceraldehydes 3-phosphate by triose phosphate
isomerase.
II Mannose
CH2OH
O
H
H
OH
Mannose is the C2 epimer of glucose.
H
OH
Mannose is a substrate for hexokinase which converts it
into Mannose 6-phosphate.
OH
OH
H
H
An enzyme similar to phosphoglucose isomerase,
phosphomannose isomerase isomerizes mannose 6phosphate into fructose-6-phosphate.
ATP
Mg2+
Hexokinase
ADP
O
-
O P O-
Fructose 6-phosphate is the substrate for PFK-1.
O
-
O
O P O CH
2 O H2C O
H
OH
OH
CH2
O
H
H
OH
H
OH
H
HO
phosphomannose isomerase
OH
OH
H
H
H
OH
III Galactose
Galactose is the C4 epimer of glucose.
CH2OH
OH
O
H
H
OH
H
H
OH
OH
H
The entry of galactose into glycolysis begins with the phosphorylation of galactose by
galactokinase to form galactose 1-phosphate.
ATP
Mg2+
Galactokinase
ADP
OH
CH2OH
O
H
H
OH
H
H
O
H
-
O
O P O
OOH
H
CH2OH
O
H
H
OH
OH
H
H
NH
N
O
O
O
O P O P O CH2 O
H
H
OOOH
H
H
OH OH
Galactose 1-phosphate is then converted into
UDP-galactose by the enzyme galactose-1phosphate uridylyltransferase with the
concurrent formation of glucose-1-phosphate
from UDP-glucose.
Galactose 1-phosphate uridylyl transferase
This enzyme has a ping pong kinetic
mechanism. UDP-glucose binds to the enzyme
O
CH2OH
first. A covalent enzyme-UMP intermediate is
NH
O H
OH
CH2OH
formed liberating glucose-1-phosphate which
H
N
O
O
O
H
OH
O H
H
O P O P O CH2
H
dissociates from the enzyme. Galactose-1H
O
O
H
OH
H
H
OOH
OH
O P OOH
phosphate then binds and reacts with the
H
H
OH O
OH
OH
H
covalent UMP-enzyme intermediate to form
UDP-galactose which then dissociates from the enzyme.
Galactosemia is the genetic disease caused by the inability of the individual to convert galactose into
glucose. The symptoms of it are mental retardation, liver damage and cataracts. This genetic disease is
caused by a deficiency of the galactose-1-phosphate uridylyltransferase enzyme. Galactosemia is
treated by a galactose free diet which reverses all of the symptoms except for the mental retardation.
H
E
EA-FP
CH2OH
O
H
H
OH
H
F
H
UDP-GAL
FB-EQ
E
The glucose 1-phosphate formed in the first step of this reaction is converted into
glucose-6-phosphate by the enzyme phosphoglucomutase.
O
O P OOOH
OH
GAL-1P
GLC-1P
UDP-GLC
Phosphoglucomutase
O
O P OO
CH2
-
H
H
OH
O
H
H
OH
OH
H
OH
O
The UDP-galactose is converted into UDP-glucose by
the enzyme UDP-glucose-4-epimerase. This enzyme
has an active site NAD+ bound and functions through
the sequential oxidation and reduction of the C4 atom.
CH2OH
NH
O H
OH
H
N
O
O
O
H
OH
O
P
O
CH
P O
H
2O
H
H
OOH
OH
H
H
OH OH
The net result of UDP-glucose uridylyltransferase and
UDP-glucose-4-epimerase is the conversion of
galactose-1-phosphate into glucose-1-phosphate.
NAD+
NADH
O
CH2OH
O
H
H
OH
O
NH
H
N
O
O
O
O P O P O CH2
O
H
H
OOOH
H
H
OH OH
H
NADH
NAD+
O
H
CH2OH
O
H
H
OH
OH
H
H
NH
N
O
O
O
O P O P O CH2
O
H
H
OOOH
H
H
OH OH