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Lecture 20
– 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
In addition to energetics -must balance redox chemistry
Redox chemistry
Glycolysis
Glucose (C6H12O6) + 6 O2
2 pyruvate + 2 (2H)
Active hydrogen 2H+ + 2e-
6 CO2 + 6 H2O
Broken down into “half pathways”
Glucose
Mitochondria
(2H) + 1/2 O2 H2O
Common carrier of (H)
NAD(P) Nicotinamide adenine dinculeotide (phosphate)(oxidized form)
O
N N
NN
O
OHHO
O-
O
O-
O
O
OHHO
CH2-O-P-O-P-CH2 N
C-N-H2
(+)
Pi
NAD+ + 2e- NADH + H+
Common carrier of (H)
NAD(P) Nicotinamide adenine dinculeotide (phosphate)(reduced form)
O
N N
NN
O
OHHO
O-
O
O-
O
O
OHHO
CH2-O-P-O-P-CH2 N
C-N-H2
Pi
H H
NADH + H+ NAD+ + 2e- Eº ‘ = 0.31 volt
Thermodynamically
Eº’ = +0.82 volt2e- + 2H+ + 1/2 O2H2O
Ease at which molecule donates
electron(s)
aka electromotive force
Eº’ = +0.31 voltNADH + H+ NAD+ + 2H+ + 2e-
NADH + H+ + 1/2 O2 NAD+ + H2O Eº’ = +1.13 volt
Convert using the Nernst Equation
Gº ‘ = -nF Eº‘ F = faraday= 23,086 calmol e- volt
Gº ‘ = -2( )131 volt)23,086 cal
mol e- volt
Gº ‘ = -56 kcal/mol
n=mol e-
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
ExampleThe G’ for hydrolysis of sugar phosphate (sugar-P)
R-OPO32- + H2O R-OH + P
sugar-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 -> CThe 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
• In the catabolic pathway...
2 ATP to activate
4 ATP + 2 NADH
Glucose
2 pyruvate
2NAD+ + 2ADP
2NADH + 2ATP
Lactate
anaerobic
NAD+
Acetyl-CoA
CO2
4 CO2
Citric acid (Krebs) cycle
O2
NADH + ADP
NAD + ATP
Respiratory chain
Ethanol + CO2
anaerobic fermentation
NAD+
Key reactions of glycolysis1. Phosphoryl transfer. A phosphoryl group
is transferred from ATP to a glycolytic intermediate or vice versa.
R-OH + ATP R-O-P-O- + ADP + H+
O
O-
Key reactions of glycolysis2. Phosphoryl shift. A phosphoryl group is
shifted within a molecule from one oxygen atom to another.
R-C-CH2-O-P-O-
O
O-H
OH
R-C-CH2-OH
H
O
-O-P-O-
O
Key reactions of glycolysis3. Isomerization. A ketose is converted to
an aldose or vice versa.
C=O
O
R
CH2OH
H-C-OH
R
C-H
Key reactions of glycolysis4. Dehydration. A molecule of water is
eliminated.
H-C-OH
H
H-C-OPO32-
H-C
COO-
C-OPO32-
+ H2O
H
COO-
Key reactions of glycolysis5. Aldol cleavage. A carbon-carbon bond is
split in a reversal of an aldol condensation.
HO-C-H
H
C=O
R
H-C-OH
R’
HO-C-H
C=OR
C
R’
H O
+
1st reaction of glycolysis (Gº’ = -4 kcal/mol)
OH1
OHO
OH
HOOH
*2
3
4
56
Glucose
OH1
O
-2O3P-O
OH
HOOH
*2
3
4
56
ATP
ADP
Glucose-6-phosphate (G6P)
Hexokinase (HK)Mg2+
First ATP utilization
Figure 17-5aConformation changes in yeast hexokinase on binding glucose. (a) Space-filling model
of a subunit of free hexokinase.
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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).
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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)
OH1
O
-2O3P-O
OH
HOOH
*2
3
4
56
Fructose-6-phosphate(F6P)
Phosphoglucoisomerase (PGI)
Glucose-6-phosphate (G6P)
CH2-OHO5
OHOH
OH
1
2
34
6-2O3P-O
isomerization of an aldose (G6P) to a ketose (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.
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7Lys
His-Glu
3rd reaction of glycolysis (Gº’ = -3.4 kcal/mol)
fructose-6-phosphate(F6P)
Phosphofructokinase (PFK)Mg2+
CH2-OHO5
OHOH
OH
1
2
34
6-2O3P-O
fructose-1,6-bisphosphate(FBP)
CH2-OPO3-2O
5
OHOH
OH
1
2
34
6-2O3P-O
ATP
ADP2nd ATP utilization
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)
Aldolase
Fructose-1,6-bisphosphate(FBP)
CH2-OPO3-2O
5
OHOH
OH
1
2
34
6-2O3P-O
C=O
H-C-O-
CH2-OH
H
PO3-2
H-C=O
H-C-OH
CH2-O- PO3-2
1(3)
2
3(1)
5 (2)
4 (1)
6 (3)
Dihydroxyacetone phosphate(DHAP)
Glyceraldehyde-3-phosphate(GAP)
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 Zn-intermediate)
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
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5th reaction of glycolysis (Gº’ = +1.83 kcal/mol)
Triose phosphate isomerase (TIM)
C=O
H-C-O-
CH2-OH
H
PO3-2H-C=O
H-C-OH
CH2-O- PO3-2
1(3)
2
3(1)
5 (2)
4 (1)
6 (3)
Dihydroxyacetone phosphate(DHAP)
Glyceraldehyde-3-phosphate(GAP)
H-C-OH
H-C-OH
CH2-O- PO3-2
enediol intermediate
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.
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6th reaction of glycolysis (Gº’ = +1.5 kcal/mol)
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
H-C=O
H-C-OH
CH2-O- PO3-2
2
1
3
Glyceraldehyde-3-phosphate(GAP)
-PO3-2 C-O
H-C-OH
CH2-O- PO3-2
2
3
O1,3-Bisphosphoglycerate (1,3-BPG)
NAD+ + Pi
NADH + H+
1
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
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7th reaction of glycolysis (Gº’ = -4.5 kcal/mol)
3-Phosphogylcerate kinase (PGK)Mg2+
-PO3-2 C-O
H-C-OH
CH2-O- PO3-2
2
3
O1,3-Bisphosphoglycerate (1,3-BPG)
ADP
ATP
1
3-Phosphoglycerate (3-PG) C-O-
H-C-OH
CH2-O- PO3-2
O
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.
Gº’ = +1.6GAP + Pi + NAD+ 1,3-BPG + NADH
3PG + ATP Gº’ = -4.5
Net reaction
Gº = -2.9
1,3-BPG + ADP
GAP + Pi + NAD+ + ADP 3PG + NADH + ATP
in kcal/mol
8th reaction of glycolysis (Gº’ = +1.06 kcal/mol)
phosphoglycerate mutase (PGM)
3-Phosphoglycerate (3-PG) C-O-
H-C-OH
CH2-O- PO3-2
O
C-O-
H-C-O-CH2-OH
PO3-2
O
2-Phosphoglycerate (2-PG)
Phosphogylcerate mutase (PGM)
Catalyzes the transfer of the high energy phosphoryl group on phosphoglycerate.
Requires catalytic amounts of 2,3-bisphosphoglycerate (2,3-BPG) -acts as the reaction primer.
Requires a phosphorylated His in the active site
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