Download Lecture 4 - UC Davis Plant Sciences

Lecture 4
Glycolysis
(Embden-Meyerhof Pathway)
Cleavage of glucose and
Substrate-level phosphorylation
of ADP to ATP
Background material
Gibbs Free Energy
•ΔG = ΔH - TΔS
•ΔG = RTlnQ – RTlnKeq
•ΔGo’ = – RTlnKeq
•ΔG = ΔGo’ + RTlnQ
Reduction potential
•ΔE = Eoxidant – Ereductant
•ΔE = – ΔG/nF
Q = [Preal]/[Rreal]
T = 298K (25oC)
Table 4
Table 3
Enzymes
•Amino acids and their reactions
•Classification
•Regulation
The “Powertrain” of Human Metabolism (Overview)
CARBOHYDRATES
Glucose
PROTEINS
LIPIDS
Amino acids
Fatty acids
Oxaloacetate
O2
Glycogen
Glucose-6-P
Pyruvate
Acetyl-CoA
CO2
Glycolysis
Lactate
Ribose-5-P
NADPH
NADH
ATP
H2O
Ketone
bodies
Cholesterol
NADH
p. 21
Aerobic Glycolysis (Overview)
Fischer projection-open chain
CHO
H
CHO
OH
H
1
HO
CH2OH
2
H
HO
H
OH
H
OH
ATP
ADP
3
HO
H
H
OH
H
OH
H
OH
H
OH
ATP
ADP
5
H
6
CH2OPO32-
CH2OPO32-
4
OH
+
OH
4 HC
CH2OPO32-
H
Haworth
projection
GLC
Ring
form
GLC-6-P
CH2OPO32-
CH2OH
OH
H
OH
OH
H
2
H
OH
ATP
H
H
OH
ADP
ATP
HPO42NAD+
6
OH
OH
G-3-P
OH
OH
H
3
OH
O
CH2OPO32-
O
H
OH
H
OH
O3POH2C
5
5
6 CH OPO 22
3
F-1,6-bisP
CH2OH
O
OH
H
OH
1
OH
O3POH2C
O
H
O
H
F-6-P
DHAP
3 CH2OH
H
4
H
O
O
3
HO
H
CH2OH
2
2
O
OH
1 CH2OPO32-
1 CH2OPO32-
H
ADP
NADH
+ H+
2 Molecules
-O
-
C
O
-O
O
C
O
10
CH3
PYR
C
OPO32-
ATP
O
ADP
CH2
PEP
H
H2O
C
O
8
9
O
OPO32-
-O
OPO32-
CH2OH
2-PGA
7
H
H
OH
CH2OPO32-
3-PGA
ATP
ADP
3,4
2,5
1,6
C
O
OH
CH2OPO32-
1,3 bisPGA
p. 25
Cellular Logic
Why add a phosphate?
CHO
CHO
H
HO
H
H
OH
H
H
HO
OH
H
OH
H
CH2OH
Cell glucose level ~ 4 - 5 mM
Blood glucose level ~ 5 mM
OH
H
OH
OH
CH2OH
Cellular Logic
Why add a phosphate?
1. Prevents reverse diffusion through GluT.
2. Prevents diffusion through plasma membrane.
3. Maintains the Glucose concentration gradient.
4. Binds to enzymes better (recognition tag).
The 1. Reaction of Glycolysis
Enzyme Class: Transferase
Specifically, phosphotransferase or “kinase”
CHO
H
HO
OH
Glucose (Glc)
NH2
ATP
H
H
OH
H
OH
N
N
O
-O P
O
-
O
Enzyme
N
O
O
P O
P O
-
O
O
O
-
H
CH2O H
N
OH
H
OH
Electrophile
(P)
Nucleophile
(-OH)
H+
p. 24,26
CHO
NH2
H
OH
HO
N
N
H
O
H
OH
H
-O P
O
O
P O
P O
-
-
N
N
O
O-
O
O
OH
O
H
CH2O-
OH
H
OH
CHO
NH2
H
HO
Glc-6-P
OH
ADP
H
O-
H
H
OH
OH
CH2O
O
P
O-
OP
O-
N
O
O
P O
N
O
OH
O
N
N
OH
H
OH
Energy coupling…
p. 26
ΔG Calculations on 1. Reaction in Glycolysis (Phosphorylation of Glucose)
Summary of Chalk Board Calculations
ΔGo’ or ΔG (kJmol-1)
Glc + Pi Î Glc-6-P +
5 mM 1 mM 0.083 mM
H2O
+13.9
+21.1
Keq
3.7x10-3
2.8x10-4
(intracellular concentrations)
ΔGo’ = –RTlnKeq
Keq = e^(-ΔGo’/RT) = e^(-13,900 Jmol-1/8.315 Jmol-1K-1 x 298 K) = 0.0037
ΔG = ΔGo’ + RTln[P]/[S]
ΔG = +13.9 kJmol-1 + (8.315 Jmol-1K-1 x 310 K) x ln[83 x10-6]/[5 x 10-3][1 x 10-3]
ΔG = +13.9 kJmol-1 + (2.578 kJmol-1) x 2.81
ΔG = +13.9 + 7.2 = +21.1 kJmol-1
(intracellular conditions are even more unfavorable than standard conditions
for the reaction to proceed as desired)
Q: How to drive glucose phosphorylation forward despite large positive ΔG?
A: Couple to much more favorable reaction (larger negative ΔG) such as to
the hydrolysis of ATP!!
ΔGo’ or ΔG (kJmol-1)
ATP
+
H2O
Î
ADP
+
Pi
-30.5
-46.5
Keq
2.2x105
6.8x107
Intracellular [ATP]/[ADP][Pi] = 500 or higher (“phosphorylation potential” )
ΔG = ΔGo’ + RTln[P]/[S]
ΔG = - 30.5 + RTln 1/500
ΔG = - 30.5 + (- 15.4) = - 46.5 kJmol-1
Combination (coupling) of both reactions via an enzyme (hexokinase):
ΔGo’ or ΔG (kJmol-1)
Keq
Glc + Pi Î Glc-6-P
Intracellular conditions
+
H2O
+13.9
+21.1
3.7x10-3
2.8x10-4
ATP + H2O Î ADP
Intracellular conditions
+
Pi
-30.5
-46.5
2.2x105
6.8x107
-16.6
-25.4
8.1x102
1.9x104
Glc + ATP Î Glc-6-P
Intracellular conditions
+
ADP
Note:
Coupling of a reaction to ATP hydrolysis can shift its Keq up to 108 –fold !!
(2.8x10-4 Î 1.9x104)
Hexokinase (induced fit)
Isoenzymes: catalyze the same reaction but differ in properties
(Liver)
Aerobic Glycolysis (Overview)
Fischer projection-open chain
CHO
H
CHO
OH
H
1
HO
CH2OH
2
H
HO
H
OH
H
OH
ATP
ADP
3
HO
H
H
OH
H
OH
H
OH
H
OH
ATP
ADP
5
H
6
CH2OPO32-
CH2OPO32-
4
OH
+
OH
4 HC
CH2OPO32-
H
Haworth
projection
GLC
Ring
form
GLC-6-P
CH2OPO32-
CH2OH
OH
H
OH
OH
H
2
H
OH
ATP
H
H
OH
ADP
ATP
HPO42NAD+
6
OH
OH
G-3-P
OH
OH
H
3
OH
O
CH2OPO32-
O
H
OH
H
OH
O3POH2C
5
5
6 CH OPO 22
3
F-1,6-bisP
CH2OH
O
OH
H
OH
1
OH
O3POH2C
O
H
O
H
F-6-P
DHAP
3 CH2OH
H
4
H
O
O
3
HO
H
CH2OH
2
2
O
OH
1 CH2OPO32-
1 CH2OPO32-
H
ADP
NADH
+ H+
2 Molecules
-O
-
C
O
-O
O
C
O
10
CH3
PYR
C
OPO32-
ATP
O
ADP
CH2
PEP
H
H2O
C
O
8
9
O
OPO32-
-O
OPO32-
CH2OH
2-PGA
7
H
H
OH
CH2OPO32-
3-PGA
ATP
ADP
3,4
2,5
1,6
C
O
OH
CH2OPO32-
1,3 bisPGA
p. 25
Fischer projection-open chain
CHO
H
OH
H
1
HO
CH2OH
CHO
2
H
H
OH
H
OH
HO
ATP
O
OH
ADP
CH2OH
HO
H
H
H
OH
H
OH
H
OH
H
OH
CH2OPO32-
CH2OPO32-
Haworth
projection
Ring
form
GLC
GLC-6-P
Isomerization
F-6-P
Reaction 2:
Phosphogluco isomerase or Glucose-6-P ketolisomerase (see p 24)
ΔGo’ = 1.67 kJ/mol
ΔG = - 2.92 kJ/mol
p. 25
CH2OH
1 CH2OPO322
O
3
HO
1 CH2OPO32-
H
H
OH
H
OH
HO
ATP
ADP
H
H
CH2OPO32-
3
4
5
6
2
O
O
4
3 CH2OH
H
OH
+
OH
4 HC
CH2OPO32-
DHAP
H
5
5
O
OH
G-3-P
Phosphorylation
F-6-P
F-1,6-bisP
6 CH OPO 22
3
Reaction 3: Phosphofructokinase-1 (PFK-1) or
ATP:Fructose-6-P 1-phosphotransferase
ΔGo’ = -14.2 kJ/mol
ΔG erythrocyte = -18.8 kJ/mol
p. 25
CH2OH
1 CH2OPO322
O
3
HO
H
H
OH
H
OH
CH2OPO32-
F-6-P
1 CH2OPO32-
HO
ATP
ADP
H
H
3
4
5
6
2
O
O
4
3 CH2OH
H
OH
Cleavage
F-1,6-bisP
H
5
+
4 HC
OH
CH2OPO32-
DHAP
5
O
OH
G-3-P
6 CH OPO 22
3
Reaction 4: Aldolase or Fructose-1,6-BisP glyceraldehyde-3-P lyase
p. 25
ΔGo’ = +23.9 kJ/mol
ΔG = -0.23 kJ/mol
Aldol Cleavage in Glycolysis
(Reaction No. 4)
CH2OP
βC
HO
H
α
O
C
H
C
O
Rest
HO
CH2OP
C
O
C
H
H
H
H
C
Rest
O
Aldol Cleavage in Glycolysis
Requirements for cleavage: C-OH must be β to carbonyl carbon
R1
R1
H
C
R2
R5
αC
β
C
O
C
R5
O
+
R3
O
R2
C
R3
C
O
R4
H
H
R4
R1
R2
C
O
C
R3
H
p. 27
Reverse Reaction (Aldol Condensation)
Condensation
Requirements for condensation: H on C that is α to carbonyl carbon on substrate 1 (C-H acidic)
and need for carbonyl group on substrate 2
Resonance stabilized
Substrate 1
R1
R2
H
C
O
C
R3
R1
R1
R2
C
O
C
R3
R2
C
O
C
R3
H
R1
H
R5
C
C
O
R2
C
R3
R5
C
OH
O
R4
Substrate 2
R4
p. 27
F-1,6-BP
DHAP
G3P
How can we force
this reaction to go
forward??
Aldolase
ΔGo’= + 23.9 kJ/mol
Le Chatelier's
Principle
ΔGo’= - 14.2 kJ/mol
ΔG = - 18.8 kJ/mol
PFK-1
F6P
Common Intermediate
F-1,6-BP
DHAP
G3P
Aldolase
ΔGo’= + 23.9 kJ/mol
ΔGo’= - 14.2 kJ/mol
ΔG = - 18.8 kJ/mol
Phosphogluco isomerase
ΔGo’= + 1.7 kJ/mol
ΔG = - 2.9 kJ/mol
Hexokinase
PFK-1
F6P
F-1,6-BP
ΔGo’= - 16.6 kJ/mol
ΔG = - 24.8 kJ/mol
DHAP
G3P
Aldolase
ΔGo’= + 23.9 kJ/mol
ΔGo’= - 16.6 kJ/mol
ΔG = - 24.8 kJ/mol
ΔGo’= - 14.2 kJ/mol
ΔG = - 18.8 kJ/mol
Hexokinase
PFK-1
F6P
F-1,6-BP
DHAP
G3P
Aldolase
ΔGo’= + 23.9 kJ/mol
ΔG = - 0.23 kJ/mol
“ripple effect”
Pyruvate
Kinase
ΔGo’= - 31.7 kJ/mol
ΔG = - 23.0 kJ/mol
CH2OH
1 CH2OPO322
O
3
HO
H
H
OH
H
OH
CH2OPO32-
F-6-P
1 CH2OPO32-
HO
ATP
ADP
H
H
3
4
5
6
2
O
O
4
3 CH2OH
H
OH
+
OH
4 HC
CH2OPO32-
F-1,6-bisP
DHAP
H
5
5
O
OH
G-3-P
6 CH OPO 22
3
Reaction 5:
Triose phosphate isomerase (ketolisomerase)
For first 5 rxns, the total ΔGo’ = +2.2 kJ/mol ΔG = -53.4 kJ/mol
p. 25
4 HC
H
5
O
OH
G-3-P
Reaction 6: The Big Deal!
6 CH OPO 22
3
23
HPO42+
NAD
6
Glyceraldehyde-3-P dehydrogenase
or
NADH
+ H+
Glyceraldehyde-3-P NAD+ oxidoreductase
(phosphorylating)
OPO32-
H
3,4
2,5
1,6
C
O
OH
CH2OPO32-
1,3 bisPGA
p. 25
The Mechanism of Reaction 6
Role of Thioesters in
Energy Transduction
Disclaimer: Do NOT memorize mechanism, understand what happens here!
Esters versus Thioesters
δ−
δ−
O
R1
C
O
O
R2
Ester
Resonance
stabilization
R1
C
O
δ−
R2
δ−
O
R1
C
No resonance stabilization of thioesters
(more “strained” molecules)
S
Thioester
R2
Therefore, ΔGo’ of thioester hydrolysis
is highly negative (about –30 kJ/mol).
See Table 4.
Mechanism of Glyceraldehyde-3-P Dehydrogenase
CH2OPO3 2CHOH
H C O-
SH
HC
H
O
OH
B
+
NAD+
+
S
NAD+
BH
Enzyme
CH2OPO32-
Glyceraldehyde-3-P
CH2OPO32CHOH
Hydride
removal
to NAD+
H C O-
S
+
NAD+
HB
p. 29
CH2OPO3 2-
CH2OPO32-
CHOH
CHOH
C O
C O
O
+ S
NAD+
+ S
HB
HB
-O P OH
+
O-
NAD
NADH
Thioester-linked
substrate
SH
2O3PO
C
H
NADH
NAD+
O
OH
+
B
Enzyme
+
H+
CH2OPO32-
1,3-Bisphosphoglycerate
Redox reaction
p. 29
NAD(P)H: Safe and Soluble Carrier of “Hydrogen”
From vitamin B (niacin)
H
2H
O
C
H+
C
Hydride acceptor/donor
N
H O
NH2
(Nicotinamide)
+
H
NH2
..
N
R
O
-O
P
O
O
Ribose
H
H
OH
H
OH
H
NH2
N
N
O
N
O
P
O
Adenine
N
O
H
O-
PPi
NADH or NADPH
H
H
H
OH
OX
NAD+ or NADP+
Ribose
Dehydrogenases
p. 28
ΔG/ΔE Calculations on 6. Reaction in Glycolysis
Summary of Chalk Board Calculations
Glyceraldehyde-3-P + Pi + NAD+ Î 1,3-Bisphoshoglycerate + NADH + H+
Can be formally written as two reactions (coupled by enzyme):
I. Glyceraldehyde-3-P + H2O + NAD+ Î 3-Phoshoglycerate + NADH + H+
II. 3-Phoshoglycerate + Pi Î 1,3-Bisphoshoglycerate + H2O
Compare ΔGo’ of both reactions
I. Glyceraldehyde-3-P + H2O + NAD+ Î 3-Phoshoglycerate + NADH + H+
Similar to oxidation of acetaldehyde to acetate (see Table 3, reactions 3 and 12)
Acetaldehyde + H2O + NAD+ Î Acetate + NADH + H+
ΔGo’ = -nFΔEo
ΔEo = EoOxidant – EoReductant
ΔEo = EoNAD+ – EoAcetaldehyde
ΔEo = - 0.32V – (-0.58V)
ΔEo = + 0.26V
ΔGo’ = - 2 (electrons) x 96.5 kJmol-1V-1 x 0.26V
ΔGo’ = - 50.2 kJmol-1
II. 3-Phoshoglycerate + Pi Î 1,3-Bisphoshoglycerate + H2O
See Table 4 for ΔGo’ of
Hydrolysis of 1,3-Bisphosphoglycerate (ΔGo’ = - 49.6 kJmol-1)
or
Formation of 1,3-Bisphosphoglycerate (ΔGo’ = + 49.6 kJmol-1)
Conclusion:
In a first approximation (because we are only looking at ΔGo’ values),
the oxidation of glyceraldehyde-3-P to 3-phosphoglycerate yields about
the same amount of energy (-50.2 kJmol-1) as is required to produce
1,3-bisphosphoglycerate from 3-phosphoglycerate and Pi (+49.6 kJmol-1).
Again, these two reactions do not occur in isolation but are coupled or
combined by the enzyme Glyceraldehyde-3-P Dehydrogenase.
Therefore, the ΔGo’ (and in fact ΔG) of the overall reaction is close to zero.