Lecture 20
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Exam 2 on Monday, Quiz next Friday
Links for glycolysis
http://www.johnkyrk.com/glycolysis.html
http://www.terravivida.com/vivida/diygly/
Metabolism and thermodynamics
– Glycolysis
Redox chemistry
In addition to energetics -must balance redox
chemistry
6 CO2 + 6 H2O
Glucose (C6H12O6) + 6 O2
Broken down into “half pathways”
Glycolysis
Glucose
Active hydrogen 2H+ + 2e-
2 pyruvate + 2 (2H)
Mitochondria
(2H) + 1/2 O2
H2O
Common carrier of (H)
O
N
N
O
N
N
O
HO
C-N-H2
O
CH2-O-P-O-P-CH2
O
O- OOH
HO
N(+)
OH
Pi
NAD(P) Nicotinamide adenine dinculeotide (phosphate)
(oxidized form)
NAD+ + 2e-
NADH + H+
Common carrier of (H)
H
H
N
N
O
N
N
O
HO
C-N-H2
O
CH2-O-P-O-P-CH2
O
O- OOH
O
HO
N
OH
Pi
NAD(P) Nicotinamide adenine dinculeotide (phosphate)
(reduced form)
NADH + H+
NAD+ + 2e-
Eº ‘ = 0.31 volt
Thermodynamically
2e- + 2H+ + 1/2 O2
NADH + H+
H2O
NAD+ + 2H+ + 2e-
NADH + H+ + 1/2 O2
Eº’ = +0.82 volt
Eº’ = +0.31 volt
NAD+ + H2O Eº’ = +1.13 volt
Convert using the Nernst Equation
Ease at which molecule
donates electron(s)
Gº ‘ = -nF Eº‘ F = faraday= 23,086 cal
aka electromotive force
n=mol e-
Gº ‘ = -2(
23,086 cal
mol  e-  volt
mol  e-  volt
)131 volt)
Gº ‘ = -56 kcal/mol
ATP and NAD(P)H
So in metabolism, ATP formed in reaction sequences where
Gº‘ > Gº‘ hydrolysis of ATP (catabolism)
Used to drive reaction with Gº‘ < Gº‘ hydrolysis (<0)
NAD(P)H production and ATP production are usually coupled
ATP and NAD(P)H are coenzymes and therefore need to be
recycled.
Thermodynamics and
Metabolism
• Standard free energy A + B <-> C + D
•
Go’ =-RT ln([C][D]/[A][B])
•
Go’ = -RT ln Keq
•
Go’ < 0 (Keq>1.0) Spontaneous forward rxn
•
Go’ = 0 (Keq=1.0) Equilibrium
•
Go’ > 0 (Keq <1.0) Rxn requires input of energy
Example
The G’ for hydrolysis of sugar phosphate (sugar-P)
R-OPO32- + H2O
sugar-P
R-OH + P
free sugar
is -6.2 kcal/mol in a hypothetical, cell in which steady-state conc of sugar-P, free
sugar, and Pi are 10-3 M, 2 X 10-4M, and 5 X 10-2M, respectively. What is G°’
for the reaction?
Steady-state is a nonequilibrium situation that prevails because of a balance between reactions that supply and remove these substances.
The initial conditions are not at equilibrium so we can assume the reaction will
proceed until it reaches equilibrium
G’ = G°’ + RT ln ([sugar][Pi]/[sugar-P])
-6.2 kcal/mol = G°’ + (1.98 X 10-3 kcal/deg mol)(298 deg)(2.3)log ([2 X 10-4M][5 X
10-2M]/[10-3 M])
G°’ = - 6.2 kcal/mol + 2.7 kcal/mol = -3.5 kcal/mol
Metabolic Pathways are not at
Equilibrium
• Metabolic pathways are not at equilibrium
A <-> B
• Instead pathways are at steady state.
A -> B -> C
The rate of formation of B = rate of utilization of B.
Maintains concentration of B at constant level.
All pathway intermediates are in steady state.
Concentration of intermediates remains constant
even as flux changes.
Glycolysis (Embden-Meyerhof-Parnas Pathway)
•
Central pathway in glucose metabolism
•
•
•
•
Present in al plants, animals, and bacteria
Source of ATP, reducing equivalents
Source of sugars
Glucose
+
In the catabolic pathway...
2NAD + 2ADP
2NADH + 2ATP
2 ATP to activate
2 pyruvate
anaerobic
fermentation
NAD+
anaerobic
4 ATP
+ 2 NADH
NAD+
Acetyl-CoA
Lactate
Ethanol
+ CO2
CO2
Citric acid
(Krebs)
cycle
4 CO2
NADH
+ ADP
O2
Respiratory
chain
NAD +
ATP
Key reactions of glycolysis
1. Phosphoryl transfer. A phosphoryl group
is transferred from ATP to a glycolytic
intermediate or vice versa.
O
R-OH + ATP
R-O-P-O- + ADP + H+
O-
Key reactions of glycolysis
2. Phosphoryl shift. A phosphoryl group is
shifted within a molecule from one oxygen
atom to another.
O
O
OH
R-C-CH2-O-P-OH
O-
-O-P-O-
O
R-C-CH2-OH
H
Key reactions of glycolysis
3. Isomerization. A ketose is converted to
an aldose or vice versa.
O
CH2OH
C=O
R
C-H
H-C-OH
R
Key reactions of glycolysis
4. Dehydration. A molecule of water is
eliminated.
COOH-C-OPO32H-C-OH
H
COOC-OPO32H-C
H
+ H2O
Key reactions of glycolysis
5. Aldol cleavage. A carbon-carbon bond is
split in a reversal of an aldol
condensation.
R
C=O
HO-C-H
H-C-OH
R’
R
C=O
H
HO-C-H
H
+
O
C
R’
1st reaction of glycolysis (Gº’ = -4 kcal/mol)
HO
O
5
4

6
1
OH
*

2
HO
3
OH
Glucose
OH
ATP
Hexokinase (HK)
Mg2+
-2O
3P-O
4
ADP
6
O
5
OH
2
HO
First ATP utilization
3
OH

1
*

OH
Glucose-6-phosphate
(G6P)
Page 586
Figure 17-5a Conformation changes in yeast
hexokinase on binding glucose. (a) Space-filling model
of a subunit of free hexokinase.
Page 586
Figure 17-5bConformation changes in yeast
hexokinase on
binding glucose. (b) Space-filling model of a subunit of
free hexokinase in complex with glucose (purple).
Mechanism by induced fit
The two lobes that form the active site cleft move to engulf
the glucose and exclude water from the active site.
This also causes catalysis by proximity.
Needs Mg2+ ATP complex for activity (free ATP is an
inhibitor of the reaction)
2nd reaction of glycolysis (Gº’ = +0.4 kcal/mol)
-2O
3P-O

6
O
5
1
OH
4
*

2
HO
Glucose-6-phosphate
(G6P)
3
OH
OH
isomerization of an aldose
(G6P) to a ketose (F6P).
Phosphoglucoisomerase
(PGI)
-2O
3P-O
1
O
6
5
OH
4
3
OH
CH2-OH
2
OH
Fructose-6-phosphate
(F6P)
Phosphoglucoisomerase: mechanism
Reaction 2 is the isomerization of an aldose (G6P) to a
ketose (F6P).
Step 1: substrate binding
Step 2: an acid (Lys side chain) catalyzes ring opening
Step 3: A base (imidazole portion of His-Glu dyad, removes the
acidic proton from C2 to form the cis-enediolate
intermediate. The proton is acidic because it is  to a
carbon group.
Step 4: Proton is transferred to C1.
Step 5: Ring closure to form the product.
Lys
Page 587
His-Glu
3rd reaction of glycolysis (Gº’ = -3.4 kcal/mol)
-2O
3P-O
1
O
6
5
CH2-OH
2
OH
4
3
OH
fructose-6-phosphate
(F6P)
OH
ATP
Phosphofructokinase
(PFK)
Mg2+
-2O
3P-O
ADP
1
O
6
5
OH
4
3
OH
2nd ATP utilization
CH2-OPO3-2
2
OH
fructose-1,6-bisphosphate
(FBP)
Phosphofructokinase: mechanism
Reaction 3 is the phosphorylation of C1 of F6P
Nucleophilic attack by the C1-OH group of F6P on Mg2+-ATP.
PFK reaction is the rate limiting step in glycolysis.
The activity is enhanced allosterically by AMP(activator) and
inhibited by ATP and citrate (inhibitors).
4th reaction of glycolysis (Gº’ = +5.73 kcal/mol)
-2O
3P-O
1
O
6
5
OH
4
3
OH
CH2-OPO3-2
2
OH
Fructose-1,6-bisphosphate
(FBP)
Aldolase
H
4 (1)
H-C=O
5 (2)
H-C-OH
6 (3)
CH2-O-PO3-2
Glyceraldehyde-3-phosphate
(GAP)
1(3)
H-C-O-PO3-2
2
3(1)
C=O
CH2-OH
Dihydroxyacetone phosphate
(DHAP)
Aldolase
Catalyzes the cleavage of FBP to form 2 trioses, GAP and
DHAP.
Reaction proceeds via an aldo cleavage (retro aldol
condensation).
There are two mechanistic classes of aldolases: Class I
(animals and plants) and Class II (fungi, algae, bacteria) proceeds through a Zn intermediate (p. 591 for Znintermediate)
Aldolase
In the Class I enzyme the reaction occurs as follows:
Step 1: substrate binidng
Step 2: reaction of the FBP carbonyl group with the side chain
amino group of Lys (Schiff base)
Step 3: C3-C4 bond cleavage resulting in the enamine
formation and release of GAP.
Step 4: Protonation of the enamine to an iminium cation
Step 5: hydrolysis of the iminium cation to release DHAP
Page 590
5th reaction of glycolysis (Gº’ = +1.83 kcal/mol)
H
4 (1)
H-C=O
1(3)
H-C-O-PO3-2
5 (2)
H-C-OH
6 (3)
2
CH2-O-PO3-2
Glyceraldehyde3-phosphate
(GAP)
Triose
phosphate
isomerase
(TIM)
H-C-OH
H-C-OH
CH2-O- PO3-2
enediol intermediate
3(1)
C=O
CH2-OH
Dihydroxyacetone
phosphate
(DHAP)
Triose phosphate isomerase (TIM)
Only GAP continues on the glycolytic pathway and TIM
catalyzes the interconversion of DHAP to GAP
Mechanism is through a general acid-base catalysis
Final reaction of the first stage of glycolysis.
Invested 2 mol of ATP to yield 2 mol of GAP.
Page 593
6th reaction of glycolysis (Gº’ = +1.5 kcal/mol)
1
Glyceraldehyde-3-phosphate H-C=O
2
(GAP)
H-C-OH
CH2-O- PO3-2
3
Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH)
NAD+ + Pi
NADH + H+
O
1,3-Bisphosphoglycerate
(1,3-BPG)
1
C-O -PO3-2
2
H-C-OH
3
CH2-O-PO3-2
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Tetramer (4 subunits)
Catalyzes the oxidation and phosphorylation of GAP by NAD+
and Pi
Used several experiments to decipher the reaction mechanism
1. GAPDH inactivated by carboxymethylcysteine-suggests
that GAPDH has active site Cys
2. GAPDH quantitatively transfers 3H from C1 of GAP to
NAD+- this is a direct hydride transfer.
3. Catalyzes the exchange of 32P and an analog acetyl
phosphate-reaction proceeds through an acyl intermediate
Page 596
7th reaction of glycolysis (Gº’ = -4.5 kcal/mol)
O
1
1,3-Bisphosphoglycerate
C-O -PO3-2
2
(1,3-BPG)
H-C-OH
3
CH2-O- PO3-2
ADP
3-Phosphogylcerate kinase
(PGK)
Mg2+
ATP
O
3-Phosphoglycerate (3-PG)
C-OH-C-OH
CH2-O-PO3-2
Phosphoglycerate kinase (PK)
First ATP generating step of glycolysis
nucleophilic attack
Phosphoglycerate kinase (PK)
Although the preceeding reaction (oxidation of GAP) is
endergonic (energetically unfavorable), when coupled with
the PK catalyzed reaction, it is highly favorable.
in kcal/mol
GAP + Pi + NAD+
1,3-BPG + ADP
1,3-BPG + NADH Gº’ = +1.6
Gº’ = -4.5
3PG + ATP
GAP + Pi + NAD+ + ADP
3PG + NADH + ATP Gº = -2.9
Net reaction
8th reaction of glycolysis (Gº’ = +1.06 kcal/mol)
O
3-Phosphoglycerate (3-PG)
C-OH-C-OH
CH2-O- PO3-2
phosphoglycerate mutase
(PGM)
O
2-Phosphoglycerate (2-PG)
C-OH-C-O- PO3-2
CH2-OH
Phosphogylcerate mutase (PGM)
Catalyzes the transfer of the high energy phosphoryl group on
phosphoglycerate.
Requires catalytic amounts of 2,3-bisphosphoglycerate (2,3BPG) -acts as the reaction primer.
Requires a phosphorylated His in the active site
Page 599