CH3C ~ S-CoA
ENERGY YIELD
O
4 Oxaloacetate
NADH + H+
Citrate 6
4 Malate
3 ATP
3 ATP
4 Fumarate
1 ATP
2 ATP
3 ATP
FADH2
Isocitrate 6
CO+2 H+
NADH
a-ketoglutarate 5
Total = 12 ATP
4 Succinate
CO2+ H+
NADH
Succinyl-CoA 4
GTP
Aconitase
CH2-COO
Prochirality
Symmetrical
HO-C-COO
CH2-COO
HO
CH2COO
CH2COO
C
C
COO
CH2-COO
OOC
OH
CH2COO
Ogston’s 3 Point Attachment Theory
Ogston’s Three-point Attachment Theory of Aconitase
Test this yourself
Middle
Index
Right Hand
Thumb
Only your right hand will fit the pattern. Therefore,
only one of the isomers of citrate will fit on the
enzyme surface in the correct orientation
Glyoxysomes
CH3C ~ S-CoA
Oxaloacetate
Succinate
COOMitochondria
H-C-COO
..
Citrate
CHO
Malate
COO
Glyoxylate
CH2
H
H-C-COO
Starch
Malate Synthase
COOCH2
(germinating plant seeds)
O
(plant organelles)
Fumarate
Glyoxylate Cycle
HO-C-COO
H
Isocitrate
Isocitrate Lyase
CO2
a-ketoglutarate
CO2
Succinyl-CoA
All 6 carbons are
preserved
Bypass
Glyoxysome
Mitochondria
Aspartate
a-Ketoglutarate
Glutamate
Oxaloacetate
Aspartate
a-Ketoglutarate
Glutamate
Oxaloacetate
NADH
Acetyl-CoA
HS-CoA
NAD+
Malate
Citrate
Isocitrate
Isocitrate lyase
Glyoxylate
Malate synthase
Succinate
Fumarate
FADH2
FAD
Succinate
Acetyl-CoA
HS-CoA
NAD+
Malate
P 625
NADH
OAA
Gluconeogenesis
Starch
Summary of Reactions
Substrate or Cofactor
Energy
Citrate synthase
Acetyl CoA, OAA
Aconitase
( H2O)
Isocitrate dehydrogenase
releases CO2
NAD+
3 ATP
a-Ketoglutarate dehydrogenase
NAD+
3 ATP
Succinyl-CoA synthetase
GDP, Pi
1 ATP
Succinate dehydrogenase
FAD
2 ATP
Fumarase
(H2O)
Malate dehydrogenase
NAD+
Feature
releases CO2
GTP
3 ATP
TOTAL
12 ATP
Table 1. Summary of Enzymes and Specific cofactor or products in the Krebs Cycle
Citrate  isocitrate
isocitrate + NAD+  a-ketoglutarate + NADH + H+ + CO2
a-ketoglutarate + HS-CoA + NAD+  succinyl CoA + NADH + H+ + CO2
succinyl CoA + GDP + Pi  succinate + HS-CoA + GTP
succinate + FAD  fumarate + FADH2
fumarate + H2O  malate
malate + NAD+  oxaloacetate + NADH + H+
Citrate + 3NAD+ + FAD + GDP + Pi + H2O
[1]
+
Oxaloacetate + 3NADH + 3H + FADH2 + 2CO2 + GTP
6
4
2
9 ATPs
2 ATPs
1 ATP
Where do we get all that energy?
Citrate + 3NAD+ + FAD + GDP + Pi + H2O
[1]
Oxaloacetate + 3NADH + 3H+ + FADH2 + 2CO2 + GTP
1. How energetic is citrate?
6 Carbons = 3 Cycle Turns
= 3 x 12 ATP per cycle
= 36 ATPs
2. How energetic is oxaloacetate
4 Carbons = 2 Cycle turns
to Citrate
= 24 ATPs
3. How energetic is malate
4 Carbons = 2 Cycle turns + 1 NADH= 27 ATPs
Regulation of the Kreb’s Cycle
Pyruvate Dehydrogenase complex
Pyruvate + TPP  Acetal-TPP + CO2
Acetal-TPP + S-S  Ac-S ^ SH + TPP
Ac-S ^ SH + HS-CoA  AcS-CoA + HS ^ SH
HS ^ SH + FAD  S-S + FADH2
FADH2 + NAD+  FAD + NADH + H+
Pyruvate + HS-CoA + NAD+  Acetyl-CoA + NADH + H+
Regulators-Activators
Regulators- Inhibitors
and AMP
Fatty acids and ATP
Key Regulatory Points:
1. Pyruvate dehydrogenase Complex
Inhibited by NADH and Acetyl-CoA
NADH
[NAD+]
Acetyl-CoA
HS-CoA
High NADH means that the cell is experiencing a
surplus of oxidative substrates and should not produce
more. Carbon flow should be redirected towards synthesis.
High Acetyl-CoA means that carbon flow into the Krebs
cycle is abundant and should be shut down and rechanneled
towards biosynthesis
Mechanism:
Text p621
1. Competitive Inhibition
NADH and acetyl-CoA reverse the pyruvate dehydrogenase
reaction by competing with NAD+ and HS-CoA
2. Covalent Modification (second level regulation)
E-1 subunits of PDH complex is subject to phosphorylation
TPP
Active
FAD
HPO4=
1
2
Insulin
3
E1-OH
PDH
phosphatase
H2O
ATP
PDH
kinase
E1-OPO3
Inactive
ADP
Epinephrine
Glucagon
Cyclic-AMP
protein kinase
ATP
Regulation of Cycle Enzymes
Enzyme
Citrate Synthase
Aconitase
Isocitrate dehydrogenase
a-Ketoglutarate dehydrogenase
Succinyl-CoA Synthase
Succinate dehydrogenase
Fumarase
Malate dehydrogenase
Go’
(kJ/mol)
-31.5
~5
-21
-33
-2.1
+6
-3.4
+29.7
All regulatory enzymes occur in the first half of the cycle
Regulation of the Citric Acid Cycle
Primary modes:
1. Substrate availability (key enzymes are subsaturated)
Allostery is not a primary mode
2. Product inhibition
3. Feedback inhibition (competitive)
Key regulators:
1. Acetyl-CoA (controls citrate synthase)
2. OAA (controls citrate synthase, regulated by NADH)
3. NADH (controls citrate synthase, isocitrate dehydrogenase
4. Calcium (stimulates NADH production)
Equilibria to Consider
O2 consumption
NADH oxidation
ATP production
Tightly coupled: affect one is to affect all
Malate + NAD+
Controls NADH and is
controlled by
NADH
OAA + NADH
A working muscle will increase
respiration and oxidize NADH. This
stimulates OAA synthesis which
stimulates citrate synthase and
isocitrate dehydrogenase reactions.
Malate + NAD+
OAA + NADH
Substrate limited
K = [OAA][NADH]
[Malate][NAD+]
K [Malate][NAD+]
Citrate
Isocitrate dehydrogenase
a-Kg dehydrogenase
[OAA][NADH]
Respiration (O2)
Respiration Increases
P 621
Pyruvate Dehydrogenase
Citrate Synthase
No
Regulation
Isocitrate
Dehydrogenase
a-Ketoglutarate
Dehydrogenase