Download Pyruvate Kinase

glyceraldehyde-3-phosphate
NAD+ + Pi
Glyceraldehyde-3-phosphate
Dehydrogenase
NADH + H+
Recall that
there are
2 G3P per
glucose.
1,3-bisphosphoglycerate
ADP
Phosphoglycerate Kinase
ATP
3-phosphoglycerate
Phosphoglycerate Mutase
2-phosphoglycerate
Enolase
H2O
phosphoenolpyruvate
ADP
Pyruvate Kinase
ATP
pyruvate
Glyceraldehyde-3-phosphate
Dehydrogenase
H
O
NAD+
1C
H
2
C
OH
+ Pi
2
CH
OPO
2
3
3
glyceraldehyde3-phosphate
OPO32
+ H+ O
NADH
1C
H
C
2
OH
2
CH
OPO
2
3
3
1,3-bisphosphoglycerate
Exergonic oxidation of the aldehyde in glyceraldehyde-3phosphate, to a carboxylic acid, drives formation of an acyl
phosphate, a "high energy" bond (~P).
This is the only step in Glycolysis in which NAD+ is reduced to
NADH.
Phosphoglycerate Kinase
O
OPO32 ADP ATP O
O
1C
H 2C OH
2
3 CH2OPO3
1,3-bisphosphoglycerate
C
1
Mg
2+
H 2C OH
2
3 CH2OPO3
3-phosphoglycerate
7. Phosphorylation by Phosphoglycerate Kinase:
1,3-bisphosphoglycerate + ADP 
3-phosphoglycerate + ATP
The enzyme undergoes substrate-induced conformational
change similar to that of Hexokinase. (hinge)
Pi is transferred from substrate to ATP
Phosphoglycerate Kinase
O
OPO32 ADP ATP O
O
1C
H 2C OH
2
3 CH2OPO3
1,3-bisphosphoglycerate
C
1
Mg
2+
H 2C OH
2
3 CH2OPO3
3-phosphoglycerate
1, 3 BPG has higher phosphoryl potential that ATP such that Pi
can be transferred to ATP.
This is called substrate level phosphorylation: since Pi donor is a
substrate with high phosphoryl potential. (differs from ETC
making ATP.)
Phosphoglycerate Mutase
O
O
C
1
O
O
C
1
H 2C OH
2
3 CH2OPO3
H 2C OPO32
3 CH2OH
3-phosphoglycerate
2-phosphoglycerate
8. Isomerization by Phosphoglycerate Mutase:
3-phosphoglycerate  2-phosphoglycerate
Phosphate is shifted from the OH on C3 to the OH on C2.
isomerase enzyme that catalyzes the structural rearrangement of
isomers.
Mutase: enzyme that catalyzes the shifting of a functional group
from one position to another within the same molecule
EC 5 Isomerases
EC 5.4 Intramolecular transferases
EC 5.4.2 Phosphomutases that transfer phospho groups
EC 5.4.2.1 D-phosphoglycerate 2,3-phosphomutase
Phosphoglycerate Mutase
O
O
C
1
H 2C OH
2
3 CH2OPO3
3-phosphoglycerate
histidine
O
O
H
C
1
H 2C OPO3
3 CH2OH
2
2-phosphoglycerate
An active site histidine side-chain
participates in Pi transfer, by donating &
accepting phosphate.
The process involves a 2,3bisphosphate
intermediate.
Enz-His-Pi + 3PG < Enz-His + 2,3BPG
Enz-His + 2,3BPG < Enz-His-Pi + 2PG
2, 3 BPG Pi donor and regenerated
H3N+
COO
C
CH2
C
HN
HC
CH
NH

O
O
C
1
H 2C OPO32
2
3 CH2OPO3
2,3-bisphosphoglycerate
Enolase
O
O

C
1
H 2 C OPO32
3 CH2OH
H

O
O

C
C
OH
O
O
1
OPO32
CH2OH
C
2C
OPO32
3 CH2
2-phosphoglycerate enolate intermediate phosphoenolpyruvate
9. Dehydration by Enolase:
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.
Enolase
O
O

C
1
H 2 C OPO32
3 CH2OH
H

O
O

C
C
OH
O
O
1
OPO32
CH2OH
C
2C
OPO32
3 CH2
2-phosphoglycerate enolate intermediate phosphoenolpyruvate
9. Dehydration by Enolase:
2-phosphoglycerate  phosphoenolpyruvate + H2O
enolate anion
http://www.biochem.wisc.edu/faculty/rayment/lab/gallery_jpgs/enolase.jpg
Pyruvate Kinase
O
O
C
1
C
2
ADP ATP
O
O
C
1
OPO32
3 CH2
phosphoenolpyruvate
C
2
O
3 CH3
pyruvate
10. Phosphorylation by Pyruvate Kinase:
phosphoenolpyruvate + ADP  pyruvate + ATP
Again a substrate-level phosphorylation with PEP have a high
phosphoryl potential.
Pyruvate Kinase
O
O
C
1
C
2
ADP ATP
O
O
C
1
OPO32
3 CH2
phosphoenolpyruvate
C
2
O
3 CH3
pyruvate
10. Phosphorylation by Pyruvate Kinase:
phosphoenolpyruvate + ADP  pyruvate + ATP
Pyruvate Kinase is highly regulated also!
Allosterically regulated by ATP, if there is enough then we
don’t need to go through glycolysis to make more!
BUT it is also regulated by.. Alanine
Pyruvate Kinase
it is also regulated by.. Alanine
transaminase
So if there is a lot of alanine in the cell, it tells the cell that
there is a lot of pyruvate, therefore enough ATP!!!!
Pyruvate Kinase
Pyruvate Kinase, the last
step Glycolysis, is
controlled in liver partly
by modulation of the
amount of enzyme.
O
O
C
1
C
2
ADP ATP
O
O
C
1
OPO32
3 CH2
phosphoenolpyruvate
C
2
O
3 CH3
pyruvate
High [glucose] within liver cells causes a transcription factor
to activate transcription of the gene for Pyruvate Kinase.
This facilitates converting excess glucose to pyruvate, which
is metabolized to acetyl-CoA, the main precursor for synthesis
of fatty acids, for long term energy storage.
Pyruvate kinase
Pyruvate kinase has at least three isozymes and one of
them is liver-specific.
The liver pyruvate kinase is being regulated differently
than other tissue type.
Pyruvate Kinase
3 isozymes
ATP, AcSCoA, long chain fatty acids inhibit all isozymes
L inhibited by phosphorylation by glucagon –activated
cAMP-dependent protein kinase (low blood sugar 
cAMP)
M activated by cAMP in response to epinephrine (Gprotein system)
Regulation of pyruvate kinase
cAMP dependent
Flux through the Glycolysis pathway is regulated by control of 3
enzymes that catalyze spontaneous reactions:
Hexokinase, Phosphofructokinase & Pyruvate Kinase.
Local control of metabolism involves regulatory effects of
varied concentrations of pathway substrates or intermediates,
to benefit the cell.
Global control is for the benefit of the whole organism, & often
involves hormone-activated signal cascades.
Liver cells have major roles in metabolism, including
maintaining blood levels various of nutrients such as glucose.
Thus global control especially involves liver.
glyceraldehyde-3-phosphate
NAD+ + Pi
Glyceraldehyde-3-phosphate
Dehydrogenase
NADH + H+
Recall that
there are
2 G3P 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:
2
How many ATP ~P bonds expended? ________
How many ~P bonds of ATP produced? (Remember there
4
are two 3C fragments from glucose.) ________
2
Net production of ~P bonds of ATP per glucose: ________
http://www.youtube.com/watch?v=mmACA_eVLTE
Balance sheet for ~P bonds of ATP:
 2 ATP expended
 4 ATP produced (2 from each of two 3C fragments from
glucose)
 Net production of 2 ATP per glucose.
Glycolysis - total pathway, omitting H+:
glucose + 2 NAD+ + 2 ADP + 2 Pi 
2 pyruvate + 2 NADH + 2 ATP
In aerobic organisms:
 pyruvate produced in Glycolysis is oxidized to CO2 via Krebs
Cycle (can also be stored as fatty acids)
 NADH produced in Glycolysis & Krebs Cycle is reoxidized via
the respiratory chain, with production of much additional ATP.
Glycolysis Enzyme/Reaction
DGo'
kJ/mol
DG
kJ/mol
Hexokinase
Phosphoglucose Isomerase
-20.9
+2.2
-27.2
-1.4
Phosphofructokinase
Aldolase
Triosephosphate Isomerase
-17.2
-25.9
+22.8
-5.9
+7.9 negative
Glyceraldehyde-3-P Dehydrogenase
& Phosphoglycerate Kinase
-16.7
-1.1
Phosphoglycerate Mutase
Enolase
Pyruvate Kinase
+4.7
-3.2
-23.0
-0.6
-2.4
-13.9
net
-44.2
*Values in this table from D. Voet & J. G. Voet (2004) Biochemistry, 3rd Edition, John Wiley & Sons, New York, p. 613.
Other Sugars
Other Fates
2 Pyruvic acid
Glucose
Figure 6.8
Glycolysis, omitting H+:
glucose + 2 NAD+ + 2 ADP + 2 Pi 
2 pyruvate + 2 NADH + 2 ATP
Fermentation, from glucose to lactate:
glucose + 2 ADP + 2 Pi  2 lactate + 2 ATP
Anaerobic catabolism of glucose yields only 2 “high
energy” bonds of ATP.
What Are the Metabolic Fates of NADH and
Pyruvate Produced in Glycolysis?
NADH is energy - two possible fates:
If O2 is available:
NADH is re-oxidized in the electron transport pathway, making
ATP in oxidative phosphorylation
If O2 is not available (anaerobic conditions):
NADH is re-oxidized by lactate dehydrogenase (LDH),
providing additional NAD+ for more glycolysis
What Are the Metabolic Fates of NADH and
Pyruvate Produced in Glycolysis?
Pyruvate is also energy - two possible fates:
If O2 is available
pyruvate enters the mitochondria, where it undergoes further
breakdown
If O2 is not available (anaerobic conditions) fermentation
occurs and pyruvate undergoes reduction
Fermentation is an anaeorbic process and does not require
oxygen.
In humans, pyruvate is reduced to lactic acid during
fermentation.
What happens when oxygen is not available?
Cells turn to fermentation.
During fermentation, pyruvate formed by glycolysis is
reduced to lactate.
The reduction of pyruvate to lactate regenerates NAD+ from
NADH.
The NAD+ is free to pick up more electrons during early
steps of glycolysis
This keeps glycolysis going.
Fermentation in Human Muscle Cells
Human muscle cells can make ATP with and without oxygen
They have enough ATP to support activities such as quick sprinting for
about 5 seconds
A secondary supply of energy (creatine phosphate) can keep muscle
cells going for another 10 seconds
To keep running, your muscles must generate ATP by the anaerobic
process of fermentation
Pyruvate is reduced by NADH, producing NAD+, which keeps glycolysis
going
In human muscle cells, lactic acid is a by-product
2 Pyruvate
Glucose
LDH
2 ADP+ 2
Glycolysis
2 NAD
2 NAD
Glucose
2 Pyruvic
acid
+ 2 H
2 Lactic
acid
(a) Lactic acid fermentation
Figure 6.15a
Lactate Dehydrogenase
O
O
C
C
NADH + H+ NAD+
O
O
O
C
HC
OH
CH3
CH3
pyruvate
lactate
Skeletal muscles ferment glucose to lactate during exercise.
Lactate released to the blood may be taken up by other
tissues, or by skeletal muscle after exercise, and converted via
Lactate Dehydrogenase back to pyruvate, which may be
oxidized in Krebs Cycle or (in liver) converted to back to
glucose via gluconeogenesis
Lactate Dehydrogenase
O
O
C
C
NADH + H+ NAD+
O
O
O
C
HC
OH
CH3
CH3
pyruvate
lactate
Lactate serves as a fuel source for cardiac muscle as well
as brain neurons.
Astrocytes, which surround and protect neurons in the
brain, ferment glucose to lactate and release it.
Lactate taken up by adjacent neurons is converted to
pyruvate that is oxidized via Krebs Cycle.
Pyruvate
Decarboxylase
Alcohol
Dehydrogenase
CO2
NADH + H+ NAD+
O
O
C
C
O
CH3
pyruvate
H
O
C
CH3
acetaldehyde
H
H
C
OH
CH3
ethanol
Some anaerobic organisms metabolize pyruvate to
ethanol, which is excreted as a waste product.
NADH is converted to NAD+ in the reaction catalyzed
by Alcohol Dehydrogenase.
Advantages and Disadvantages of Fermentation
Fermentation can provide a rapid burst of ATP in
muscle cells, even when oxygen is in limited supply.
Lactate, however, is toxic to cells.
Initially, blood carries away lactate as it forms;
eventually lactate builds up, lowering cell pH, and
causing muscles to fatigue.
Oxygen debt occurs, and the liver must reconvert
lactate to pyruvate.
Efficiency of Fermentation
Two ATP produced during fermentation are
equivalent to 14.6 kcal.
Complete oxidation of glucose to CO2 and H2O
represents a yield of 686 kcal per molecule of
glucose.
Thus, fermentation is only 2.1% efficient compared
to cellular respiration.
(14.6/686) x 100 = 2.1%