Citric Acid Cycle

1
Citric Acid Cycle
2
Anabolism and Catabolism(Heterotrophs)
3
Oxidative Fuel Metabolism
Figure 17-1
4
Summary of Anaerobic Glycolysis
Glucose 2 ADP 2 Pi
2 Lactate 2 ATP 2 H2O 2 H
5
Energetics of Fermentation
Glucose gt 2 Lactate Glucose 6 O2 gt 6 CO2
6 H2O
?Go -200 kJ/mol ?Go -2866 kJ/mol
Most of the energy of glucose is still available
following glycolysis!
6
Carbon Atom Oxidation
CH2 gt CH2OH gt CO gt CO2
7
Oxidation-Reduction Reactions
SH2 NAD H2O gt S NADH H3O
SH2 Reduced Substrate S Oxidized Product NAD
Electron Acceptor FAD Electron Acceptor
8
Electron Transfer
Substrate gt NAD or FAD gt Electron Carriers gt
O2
Electron Transport Oxidative Phosphorylation
9
Electron TransportOxidative Phosphorylation
10
Oxidative Phosphorylation
11
Citric Acid Cycle
Figure 17-2
12
Amphibolic Nature of Citric Acid Cycle
13
Summary of Citric Acid Cycle
Acetyl-CoA 3 NAD FAD GDP Pi
2 CO2 3 NADH 3H FADH2 GTP CoA-SH
14
Synthesis of Acetyl-CoA
15
Sources of Acetyl-CoA

• Carbohydrates (sugars via glycolysis)
• Fats (fatty acids)
• Proteins (amino acids)

16
Oxidative Fuel Metabolism
Figure 17-1
17
Pyruvate Dehydrogenase (PDH)

• Formation of Acetyl-SCoA
• Multienzyme Complex

18
Pyruvate Dehydrogenase(Formation of Acetyl-SCoA)
Oxidative Decarboxylation
19
Pyruvate Dehydrogenase(Multienzyme Complex)

• E1 Pyruvate Dehydrogenase or Pyruvate
  Decarboxylase
• E2 Dihydrolipoyl Transacetylase
• E3 Dihydrolipoyl Dehydrogenase

20
Multienzyme Complexes

• Enhanced reaction rates
• Channeling of reaction intermediates
• Coordinate regulation

21
Electron Micrograph of E. coli Pyruvate
Dehydrogenase
Figure 17-3a
22
Structural Organization of E. coli Pyruvate
Dehydrogenase
E2 Core
E1
E3
Figure 17-4
23
Pyruvate Dehydrogenase(Mammalian Enzyme)

• E1, E2, and E3
• E3 binding protein
• Kinase (regulation)
• Phosphatase (regulation)

24
Coenzymes and Prosthetic Groups of Pyruvate
Dehydrogenase
Table 17-1
25
Thiamin Pyrophosphate
26
Lipoic Acid
27
Reduction of Lipoamide
E2
E2
Figure 17-7
28
Coenzyme A
29
NAD
30
Flavin Adenine Dinucleotide (FAD)
Figure 14-12
31
Reduction of FAD
32
Pyruvate Dehydrogenase(Formation of Acetyl-SCoA)
Oxidative Decarboxylation
33
Overall Reaction ofPyruvate Dehydrogenase
Figure 17-6
34
Mechanism of Pyruvate Dehydrogenase(Decarboxylati
on of Pyruvate)
Same mechanism as Pyruvate Decarboxylase
35
Mechanism of Decarboxylation of Pyruvate
Page 572
36
Mechanism of Pyruvate Dehydrogenase(Hydroxyethyl
Group Transfer)
37
Mechanism of Hydroxyethyl Group Transfer
Page 574
38
Mechanism of Pyruvate Dehydrogenase(Transesterifi
cation)
39
Mechanism of Transesterification
Page 574
40
Mechanism of Pyruvate Dehydrogenase(Reoxidation
of Dihydrolipoamide)
41
Mechanism of Pyruvate Dehydrogenase(Oxidation of
E3FADH2)
E3FADH2 NAD gt E3FAD NADH H
42
Mechanism of Reoxidation of Dihydrolipoamide
Page 574
43
Mechanism of Oxidation of E3FADH2
Page 574
44
A Swinging Arm Transfers Intermediates
E2
Page 575
45
Pyruvate Dehydrogenase(Formation of Acetyl-SCoA)
Oxidative Decarboxylation
46
Regulation of Pyruvate Dehydrogenase

• Product Inhibition (competitive)
• NADH
• Acetyl-SCoA
• Phosphorylation/Dephosphorylation
• PDH Kinase inactivation
• PDH Phosphatase reactivation

47
Regulation of PDH Kinase(Inactivation)

• Activators
• NADH
• Acetyl-SCoA
• Inhibitors
• Pyruvate
• ADP
• Ca2 (high Mg2)
• K

48
Regulation of PDH Phosphatase(Reactivation)

• Activators
• Mg2
• Ca2

49
Reactions of the Citric Acid Cycle
50
Enzymes of the Citric Acid Cycle

• Citrate Synthase
• Aconitase
• Isocitrate Dehydrogenase
• ?-Ketoglutarate Dehydrogenase
• Succinyl-CoA Synthetase
• Succinate Dehydrogenase
• Fumarase
• Malate Dehydrogenase

51
Citrate Synthase(citrate condensing enzyme)
?Go 31.5 kJ/mol
52
Mechanism of Citrate Synthase(Formation of
Acetyl-SCoA Enolate)
Figure 17-10 part 1
53
Mechanism of Citrate Synthase(Acetyl-CoA Attack
on Oxaloacetate)
Figure 17-10 part 2
54
Mechanism of Citrate Synthase(Hydrolysis of
Citryl-SCoA)
Figure 17-10 part 2
55
Regulation of Citrate Synthase

• Pacemaker Enzyme (rate-limiting step)
• Rate depends on availability of substrates
• Acetyl-SCoA
• Oxaloacetate

56
Aconitase
Stereospecific Addition
57
Iron-Sulfur Complex(4Fe-4S
Thought to coordinate citrate OH to facilitate
elimination
Page 605
58
Stereospecificity of Aconitase Reaction
Prochiral Substrate
Chiral Product
Page 325
59
Stereospecificity in Substrate Binding
Figure 11-2
60
NADDependentIsocitrate Dehydrogenase
Oxidative Decarboxylation NOTE CO2 from
oxaloacetate
61
Mechanism of Isocitrate Dehydrogenase(Oxidation
of Isocitrate)
Mn2 polarizes CO
Figure 17-11 part 1
62
Mechanism of Isocitrate Dehydrogenase(Decarboxyla
tion of Oxalosuccinate)
Mn2 polarizes CO
Figure 17-11 part 2
63
Mechanism of Isocitrate Dehydrogenase(Formation
of ?-Ketoglutarate)
Figure 17-11 part 2
64
Regulation of Isocitrate Dehydrogenase

• Pulls aconitase reaction
• Regulation (allosteric enzyme)
• Positive Effector ADP (energy charge)
• Negative Effector ATP (energy charge)
• Accumulation of Citrate inhibits
  Phosphofructokinase

65
Aconitase
66
?-Ketoglutarate Dehydrogenase
Oxidative Decarboxylation Mechanism similar to
PDH CO2 from oxaloacetate High energy thioester
67
a-Ketoglutarate Dehydrogenase(Multienzyme
Complex)

• E1 ?-Ketoglutarate Dehydrogenase or
  ?-Ketoglutarate Decarboxylase
• E2 Dihydrolipoyl Transsuccinylase
• E3 Dihydrolipoyl Dehydrogenase (same as E3 in
  PDH)

68
Regulation of ?-Ketoglutarate Dehydrogenase

• Inhibitors
• NADH
• Succinyl-SCoA
• Activator Ca2

69
Origin of C-atoms in CO2
Both CO2 carbon atoms derived from oxaloacetate
70
Succinyl-CoA Synthetase(Succinyl Thiokinase)
High Energy Thioester gt Phosphoanhydride
Bond Plants and Bacteria ADP Pi gt ATP
71
Citrate Synthase(citrate condensing enzyme)
Synthetase versus Synthase
72
Thermodynamics(Succinyl-SCoA Synthetase)
73
Evidence for Phosphoryl-enzyme Intermediate(Isoto
pe Exchange)
Absence of Succinyl-SCoA
Page 581
74
Mechanism of Succinyl-CoA Synthetase(Formation
of High Energy Succinyl-P)
Figure 17-12 part 1
75
Mechanism of Succinyl-CoA Synthetase(Formation
of Phosphoryl-Histidine)
Figure 17-12 part 2
76
Mechanism of Succinyl-CoA Synthetase(Phosphoryl
Group Transfer)
Figure 17-12 part 3
77
Nucleoside Diphosphate Kinase(Phosphoryl Group
Transfer)
GTP ADP gt GDP ATP
?Go 0
78
Succinate Dehydrogenase
Randomization of C-atom Labeling Membrane-Bound
Enzyme
79
MalonateInhibitor of Succinate Dehydrogenase
Competitive Inhibitor
Page 582
80
Covalent Attachment of FAD
Figure 17-13
81
Fumarase
82
Mechanism of Fumarase
Page 583
83
Malate Dehydrogenase
?Go 29.7 kJ/mol Low Oxaloacetate
84
Thermodynamics
85
Citric Acid Cycle
Figure 17-2
86
Amphibolic Nature of Citric Acid Cycle
87
GlycolysisandGluconeogenesis
88
Substrate Cycles in Glucose Metabolism
Figure 16-21
89
Reversal of Pyruvate Kinase Reaction
90
Fatty Acid Biosynthesis

• Condensation of 2-C Units
• Reversal of b-Oxidation

91
Pathway Overview
92
Comparison(Fatty Acid Biosynthesis versus
Degradation)

• Different pathway
• Different location
• Uses ACP versus CoASH
• D-hydroxyacyl group versus L-hydroxyacyl group
• Uses NADPH versus NAD and FAD
• Uses Malonyl-CoA versus Acetyl-CoA

93
Transport of Mitochondrial Acetyl-CoAintothe
Cytosol
94
Ammonium Assimilation(Biosynthetic Glutamate
Dehydrogenase)
95
Ammonium Assimilation (Glutamine Synthetase)
96
Microbial Nitrogen Acquisition(Metabolic Sources
of Organic Nitrogen)

• Glutamate (90)
• Amino Acids (90)
• Purines (50)
• Pyrimidines (50)

• Glutamine (10)
• Amino Acids
• Amino Sugars
• NAD
• PABA
• Purines (50)
• Pyrimidines (50)

97
Role of Glutamate(Nitrogen Donor)
98
Role of Glutamine(Nitrogen Donor)
99
Aspartate and Asparagine Biosynthesis
100
Glutamate and Glutamine Biosynthesis
101
Proline Biosynthesis
102
Arginine Metabolism in Microorganisms(Linear
Biosynthetic Pathway)
103
Generation of Citric Acid Cycle Intermediates
104
Pyruvate Carboxylase

• Mitochondrial Matrix

105
Pyruvate Carboxylase
Animals and Some Bacteria
106
Biotin Cofactor(CO2 Carrier)
107
Reaction Mechanism I(Dehydration/Activation of
HCO3)
108
Reaction Mechanism II(Transfer of CO2 to
Pyruvate)
109
Fates of Oxaloacetate
Regulation!
110
Regulation of Pyruvate Carboxylase

• Allosteric Activator
• Acetyl-SCoA

111
Glyoxylate Cycle

• Glyoxysome
• Plants and Some Microorganisms

112
Citrate Synthase(citrate condensing enzyme)
113
Aconitase
114
Glyoxylate Cycle Enzymes(Glyoxysome)
Plants and Some Microorganisms
115
Malate Dehydrogenase
116
Net Reaction of Glyoxylate Cycle
Net increase of one 4-carbon unit!
117
Glyoxylate Cycle and the Glyoxysome
Figure 17-18
118
Regulation of the Citric Acid Cycle
119
Amphibolic Nature of TCA Cycle
120
Products of the Citric Acid Cycle
Figure 17-14
121
ATP Production
Page 584
122
Regulatory Mechanisms

• Availability of substrates
• Acetyl-CoA
• Oxaloacetate
• Oxygen (O2)
• Need for citric acid cycle intermediates as
  biosynthetic precursors
• Demand for ATP

123
Free Energy Changes of Citric Acid Cycle Enzymes
Table 17-2
124
Regulation of Pyruvate Dehydrogenase

• Product Inhibition (competitive)
• NADH
• Acetyl-SCoA
• Phosphorylation/Dephosphorylation
• PDH Kinase inactivation
• PDH Phosphatase reactivation

125
Covalent Modification and Regulation of PDH
Figure 17-15
126
Regulation of PDH Kinase(Inactivation)

• Activators
• NADH
• Acetyl-SCoA
• Inhibitors
• Pyruvate
• ADP
• Ca2 (high Mg2)
• K

127
Regulation of PDH Phosphatase(Reactivation)

• Activators
• Mg2
• Ca2

128
Regulation of Citrate Synthase

• Pacemaker Enzyme (rate-limiting step)
• Rate depends on availability of substrates
• Acetyl-SCoA
• Oxaloacetate

129
Regulation of Isocitrate Dehydrogenase

• Pulls aconitase reaction
• Regulation (allosteric enzyme)
• Positive Effector ADP (energy charge)
• Negative Effector ATP (energy charge)
• Accumulation of Citrate inhibits
  Phosphofructokinase

130
Regulation of ?-Ketoglutarate Dehydrogenase

• Inhibitors
• NADH
• Succinyl-SCoA
• Activator Ca2

131
Regulation of the Citric Acid Cycle
Figure 17-16
132
Regulation of Central Metabolic Pathways
133
Metabolism During Exercise
Page 590