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Glycolysis: Biochemistry of Metabolism
Glycolysis: Biochemistry of Metabolism
Glycolysis
6 CH OPO 2
2
3
5
O
H
4
OH
H
OH
3
H
2
H
1
OH
OH
glucose-6-phosphate
6 CH2OH
5
H
4
OH
H
OH
H
2
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
Mg2+
OH
H
OH
3
H
2
H
1
OH
Hexokinase H
OH
glucose-6-phosphate
1. Hexokinase catalyzes:
Glucose + ATP glucose-6-P + ADP
The reaction involves nucleophilic attack of the C6
hydroxyl O of glucose on P of the terminal phosphate
of ATP.
ATP binds to the enzyme as a complex with Mg++.
NH2
ATP
adenosine triphosphate
O
P
O
P
O
O
O
P
O
CH2
adenine
OH
H
OH
ribose
6 CH2OH
5
H
4
OH
H
OH
H
2
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
Mg2+
OH
Hexokinase
H
OH
3
H
2
H
1
OH
OH
glucose-6-phosphate
6 CH2OH
Induced fit:
Glucose binding
to Hexokinase
stabilizes a
conformation
in which:
H
4
OH
H
OH
H
2
OH
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
Mg
2+
OH
Hexokinase
glucose
H
OH
3
H
1
H
2
OH
OH
glucose-6-phosphate
glucose
glucose
Hexokinase
6 CH OPO 2
2
3
5
O
H
4
OH
H
OH
3
H
2
OH
H
1
OH
6 CH OPO 2
2
3
1CH2OH
H
4
OH
HO
3 OH
Phosphoglucose Isomerase
glucose-6-phosphate
fructose-6-phosphate
Phosphofructokinase
6 CH OPO 2
2
3
1CH2OH
H
4
OH
ATP ADP
HO
3 OH
fructose-6-phosphate
6 CH OPO 2
2
3
Mg2+
1CH2OPO32
H
4
OH
HO
3 OH
fructose-1,6-bisphosphate
3. Phosphofructokinase catalyzes:
fructose-6-P + ATP fructose-1,6-bisP + ADP
This highly spontaneous reaction has a mechanism similar
to that of Hexokinase.
The Phosphofructokinase reaction is the rate-limiting step
of Glycolysis.
The enzyme is highly regulated, as will be discussed later.
1CH2OPO3
2C
HO 3C
H 4C
Aldolase
2
CH
OPO
2
3
3
OH
2C
OH
1CH2OH
2
CH
OPO
2
3
6
fructose-1,6bisphosphate
O
1C
H 2C OH
2
3 CH2OPO3
dihydroxyacetone glyceraldehyde-3phosphate
phosphate
Triosephosphate Isomerase
1CH2OPO3
2C
HO 3C
H 4C
Aldolase
2
CH
OPO
2
3
3
OH
2C
OH
1CH2OH
2
CH
OPO
2
3
6
fructose-1,6bisphosphate
O
1C
H 2C OH
2
3 CH2OPO3
dihydroxyacetone glyceraldehyde-3phosphate
phosphate
Triosephosphate Isomerase
Triosephosphate Isomerase
H
H
OH
H H
CH2OPO32
dihydroxyacetone
phosphate
OH
C
C
H H
OH
CH2OPO32
enediol
intermediate
O
C
OH
CH2OPO32
glyceraldehyde3-phosphate
OH
O
HC
CH2OPO32
CH2OPO32
proposed
enediolate
intermediate
phosphoglycolate
transition state
analog
Triosephosphate Isomerase
structure is an barrel, or
TIM barrel.
In an barrel there are
8 parallel -strands
surrounded by 8 -helices.
Short loops connect alternating
-strands & -helices.
TIM
TIM
OH
O
HC
TIM
CH2OPO32
CH2OPO32
proposed
enediolate
intermediate
phosphoglycolate
transition state
analog
Glyceraldehyde-3-phosphate
Dehydrogenase
H
NAD+
1C
OH
+ Pi
2
CH
OPO
2
3
3
glyceraldehyde3-phosphate
OPO32
+ H+ O
NADH
1C
H
OH
2
CH
OPO
2
3
3
1,3-bisphosphoglycerate
6. Glyceraldehyde-3-phosphate Dehydrogenase
catalyzes:
glyceraldehyde-3-P + NAD+ + Pi
1,3-bisphosphoglycerate + NADH + H+
Glyceraldehyde-3-phosphate
Dehydrogenase
H
NAD+
1C
OH
+ Pi
2
CH
OPO
2
3
3
glyceraldehyde3-phosphate
OPO32
+ H+ O
NADH
1C
H
OH
2
CH
OPO
2
3
3
1,3-bisphosphoglycerate
1C
H 2 C OH
2
3 CH2OPO3
glyceraldehyde-3phosphate
A cysteine thiol at the active site of Glyceraldehyde3-phosphate Dehydrogenase has a role in catalysis.
The aldehyde of glyceraldehyde-3-phosphate reacts
with the cysteine thiol to form a thiohemiacetal
intermediate.
Enz-Cys
Oxidation to a
carboxylic acid
(in a ~ thioester)
occurs, as NAD+
is reduced to
NADH.
Enz-Cys
OH
HC
CH
SH
OH
OH
CH
CH
CH2OPO32
glyceraldehyde-3phosphate
CH2OPO32
thiohemiacetal
intermediate
NAD+
NADH
Enz-Cys
OH
CH
CH2OPO32
acyl-thioester
intermediate
Pi
Enz-Cys
SH
O3PO
OH
CH
CH2OPO32
1,3-bisphosphoglycerate
O
C
+
N
NH2
2e + H
NH2
NAD+
NADH
Phosphoglycerate Kinase
O
O
1C
H 2C OH
2
3 CH2OPO3
1,3-bisphosphoglycerate
Mg
2+
H 2C OH
2
3 CH2OPO3
3-phosphoglycerate
Phosphoglycerate Mutase
O
O
C
O
C
H 2C OH
2
CH
OPO
2
3
3
H 2C OPO32
3 CH2OH
3-phosphoglycerate
2-phosphoglycerate
Phosphoglycerate Mutase
O
O
C
O
C
H 2C OH
2
CH
OPO
2
3
3
H 2C OPO32
3 CH2OH
3-phosphoglycerate
2-phosphoglycerate
O
C
H 2C OPO32
2
3 CH2OPO3
2,3-bisphosphoglycerate
Enolase
O
C
H 2 C OPO32
3 CH2OH
H
O
O
C
C
OH
O
1
OPO32
CH2OH
2C
OPO32
3 CH2
9. Enolase catalyzes:
2-phosphoglycerate phosphoenolpyruvate + H2O
This dehydration reaction is Mg++-dependent.
2 Mg++ ions interact with oxygen atoms of the substrate
carboxyl group at the active site.
The Mg++ ions help to stabilize the enolate anion
intermediate that forms when a Lys extracts H+ from C #2.
Pyruvate Kinase
O
O
C
1
C
2
ADP ATP
O
C
OPO32
3 CH2
phosphoenolpyruvate
3 CH3
pyruvate
Pyruvate Kinase
O
O
C
1
C
2
ADP ATP
OPO32
3 CH2
phosphoenolpyruvate
O
C
O
1
OH
3 CH2
enolpyruvate
3 CH3
pyruvate
glucose
ATP
Glycolysis
Hexokinase
ADP
glucose-6-phosphate
Phosphoglucose Isomerase
fructose-6-phosphate
ATP
Phosphofructokinase
ADP
fructose-1,6-bisphosphate
Aldolase
glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate
Triosephosphate
Isomerase
Glycolysis continued
glyceraldehyde-3-phosphate
NAD+ + Pi
Glyceraldehyde-3-phosphate
Dehydrogenase
NADH + H+
Glycolysis
continued.
Recall that
there are 2
GAP per
glucose.
1,3-bisphosphoglycerate
ADP
Phosphoglycerate Kinase
ATP
3-phosphoglycerate
Phosphoglycerate Mutase
2-phosphoglycerate
Enolase
H2O
phosphoenolpyruvate
ADP
Pyruvate Kinase
ATP
pyruvate
Glycolysis
Balance sheet for ~P bonds of ATP:
How many ATP ~P bonds expended? ________
2
How many ~P bonds of ATP produced? (Remember
4
there are two 3C fragments from glucose.) ________
Net production of ~P bonds of ATP per glucose:
2
________
Glyceraldehyde-3-phosphate
Dehydrogenase
H
Fermentation:
Anaerobic
organisms lack a
respiratory chain.
NAD+
1C
OH
+ Pi
2
CH
OPO
2
3
3
glyceraldehyde3-phosphate
OPO32
+ H+ O
NADH
1C
H
OH
2
CH
OPO
2
3
3
1,3-bisphosphoglycerate
Lactate Dehydrogenase
O
O
C
C
NADH + H+ NAD+
O
C
HC
OH
CH3
CH3
pyruvate
lactate
Lactate Dehydrogenase
O
O
C
C
NADH + H+ NAD+
O
C
HC
OH
CH3
CH3
pyruvate
lactate
Lactate Dehydrogenase
O
O
C
C
NADH + H+ NAD+
O
C
HC
OH
CH3
CH3
pyruvate
lactate
Pyruvate
Decarboxylase
Alcohol
Dehydrogenase
CO2
NADH + H+ NAD+
O
C
C
CH3
pyruvate
O
C
CH3
acetaldehyde
OH
CH3
ethanol
Go'
G
kJ/mol kJ/mol
Hexokinase
-20.9 -27.2
Phosphoglucose Isomerase
+2.2
-1.4
Phosphofructokinase
-17.2 -25.9
Aldolase
+22.8
-5.9
Triosephosphate Isomerase
+7.9 negative
Glyceraldehyde-3-P Dehydrogenase
-16.7
-1.1
& Phosphoglycerate Kinase
Glycolysis Enzyme/Reaction
Phosphoglycerate Mutase
Enolase
Pyruvate Kinase
+4.7
-3.2
-23.0
-0.6
-2.4
-13.9
*Values in this table from D. Voet & J. G. Voet (2004) Biochemistry, 3 rd Edition, John
Wiley & Sons, New York, p. 613.
6 CH2OH
5
H
4
OH
H
OH
H
2
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
Mg2+
OH
Hexokinase
H
OH
3
H
2
H
1
OH
OH
glucose-6-phosphate
6 CH2OH
5
Glucokinase
is a variant of
Hexokinase
found in liver.
OH
H
OH
H
2
OH
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
Mg2+
OH
Hexokinase
glucose
H
OH
3
H
1
H
2
OH
OH
glucose-6-phosphate
Glycogen
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway
Glucokinase,
with high KM
Glucose-1-P
for glucose,
allows liver to
store glucose
Pyruvate
as glycogen in
Glucose metabolism in liver.
the fed state
when
blood
[glucose] is high. catalyzes hydrolytic release of Pi
Glucose-6-phosphatase
from glucose-6-P. Thus glucose is released from the liver
to the blood as needed to maintain blood [glucose].
Pyruvate Kinase
O
ATP
O
C
3 CH3
pyruvate
Phosphofructokinase
6 CH OPO 2
2
3
1CH2OH
H
4
OH
ATP ADP
HO
3 OH
fructose-6-phosphate
6 CH OPO 2
2
3
Mg2+
1CH2OPO32
H
4
OH
HO
3 OH
fructose-1,6-bisphosphate
60
low [ATP]
PFK Activity
50
40
30
high [ATP]
20
10
0
0
0.5
1
1.5
[Fructose-6-phosphate] mM
Glycogen
Glucose-1-P
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.