Citric Acid Cycle

Figure 17-2 Citric Acid Cycle

Summary of Citric Acid Cycle Acetyl-CoA + 3 NAD + + FAD + GDP + P i 2 CO NADH + 3H + + FADH 2 + GTP + CoA-SH

Reactions of the Citric Acid Cycle

Citrate Synthase (citrate condensing enzyme) ∆G o ’ = –31.5 kJ/mol

Figure part 1 Mechanism of Citrate Synthase (Formation of Acetyl-SCoA Enolate)

Figure part 2 Mechanism of Citrate Synthase (Acetyl-CoA Attack on Oxaloacetate)

Figure part 2 Mechanism of Citrate Synthase (Hydrolysis of Citryl-SCoA)

Regulation of Citrate Synthase Pacemaker Enzyme (rate-limiting step) Rate depends on availability of substrates –Acetyl-SCoA –Oxaloacetate

Aconitase Stereospecific Addition ∆G o ’ = ~0

Iron-Sulfur Complex (4Fe-4S] Thought to coordinate citrate –OH to facilitate elimination

Page 325 Stereospecificity of Aconitase Reaction Prochiral SubstrateChiral Product

Figure 11-2 Stereospecificity in Substrate Binding

NAD + –Dependent Isocitrate Dehydrogenase Oxidative Decarboxylation NOTE: CO 2 from oxaloacetate ∆G o ’ = kJ/mol

Figure part 1 Mechanism of Isocitrate Dehydrogenase (Oxidation of Isocitrate)

Figure part 2 Mechanism of Isocitrate Dehydrogenase (Decarboxylation of Oxalosuccinate) Mn 2+ polarizes C=O

Figure part 2 Mechanism of Isocitrate Dehydrogenase (Formation of  -Ketoglutarate)

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

Accumulation of Citrate CO 2 Isocitrate dehydrogenase CO 2 Isocitrate dehydrogenase

 -Ketoglutarate Dehydrogenase Oxidative Decarboxylation Mechanism similar to PDH CO 2 from oxaloacetate High energy thioester ∆G o ’ = kJ/mol

 -Ketoglutarate Dehydrogenase (Multienzyme Complex) E 1 :  -Ketoglutarate Dehydrogenase or  -Ketoglutarate Decarboxylase E 2 : Dihydrolipoyl Transsuccinylase E 3 : Dihydrolipoyl Dehydrogenase (same as E 3 in PDH)

Regulation of  -Ketoglutarate Dehydrogenase Inhibitors –NADH –Succinyl-SCoA Activator: Ca 2+

Origin of C-atoms in CO 2 Both CO 2 carbon atoms derived from oxaloacetate

Succinyl-CoA Synthetase (Succinyl Thiokinase) High Energy Thioester —> Phosphoanhydride Bond Plants and Bacteria: ADP + P i —> ATP Randomizationn of labeled C atoms ∆G o ’ = ~0

Thermodynamics (Succinyl-SCoA Synthetase)

Page 581 Evidence for Phosphoryl-enzyme Intermediate (Isotope Exchange) Absence of Succinyl-SCoA

Figure part 1 Mechanism of Succinyl-CoA Synthetase (Formation of High Energy Succinyl-P)

Figure part 2 Mechanism of Succinyl-CoA Synthetase (Formation of Phosphoryl-Histidine)

Figure part 3 Mechanism of Succinyl-CoA Synthetase (Phosphoryl Group Transfer) Substrate-level phosphorylation

Nucleoside Diphosphate Kinase (Phosphoryl Group Transfer) GTP + ADP ——> GDP + ATP ∆G o ’ = ~0

Succinate Dehydrogenase Randomization of C-atom Labeling Membrane-Bound Enzyme ∆G o ’ = ~0

Figure Covalent Attachment of FAD

FAD used for Alkane  Alkene Reduction Potential –Affinity for electrons; Higher E, Higher Affinity –Electrons transferred from lower to higher E  E h o’ =  G o’ /nF = -(RT/nF)ln (K eq ) FAD/FADH 2 Succinate/Fumarate NAD+/NADH Isocitrate/α-Ketoglutarate Reduction Potential

Fumarase ∆G o ’ = ~0

Page 583 Mechanism of Fumarase

Malate Dehydrogenase ∆G o ’ = kJ/mol Low [Oxaloacetate]

Thermodynamics

Figure Products of the Citric Acid Cycle

Page 584 ATP Production from Products of the Central metabolic Pathway = 32 ATP NADH  2.5 ATP FADH 2  1.5 ATP

Amphibolic Nature of Citric Acid Cycle

Carbons of Glucose: 1st cycle , 4 2,5 1,6 2,5 1,6 2,5 1,6 2,5

Carbons of Glucose: 2nd cycle: Carbons 2,5: After 1½ turns: all radioactivity is CO 2

Carbons of Glucose: 2nd cycle: Carbons 1,6: After 2 turns: ¼ radioactivity in each carbon of OAA

Carbons of Glucose: 3rd cycle: Carbons 1,6: After 3 turns: ½ radioactivity is CO 2 Each turn after will lose ½ remaining radioactivity

Carbon Tracing from Glucose Glucose radiolabeled at specific Carbons –Can determine fate of individual carbons Carbons 1,6 –1 st cycle: 1, 4 of oxaloacetate –Starting at 3 rd cycle ½ radioactivity  CO 2 /cycle Carbons 2,5 –1 st cycle: 2, 3 of oxaloacetate –2 nd cycle: all eliminated as CO 2 Carbons 3,4 –All eliminated at CO 2 during Pyruvate  Acetyl-CoA

You are following the metabolism of pyruvate in which the methyl-carbon is radioactive: *CH 3 COCOOH. -assuming all the pyruvate enters the TCA cycle as Acetyl-CoA, indicate the labeling pattern and its distribution in oxaloacetate first formed by operation of the TCA cycle.

Generation of Citric Acid Cycle Intermediates

Pyruvate Carboxylase Mitochondrial Matrix

Pyruvate Carboxylase Animals and Some Bacteria

Biotin Cofactor (CO 2 Carrier)

Reaction Mechanism I (Dehydration/Activation of HCO 3 – )

Reaction Mechanism II (Transfer of CO 2 to Pyruvate)

Fates of Oxaloacetate Regulation!

Regulation of Pyruvate Carboxylase Allosteric Activator Acetyl-SCoA

Glyoxylate Cycle Glyoxysome Plants and Some Microorganisms

Citrate Synthase (citrate condensing enzyme)

Aconitase

Glyoxylate Cycle Enzymes Plants and Some Microorganisms

Malate Dehydrogenase

Net Reaction of Glyoxylate Cycle Net increase of one 4-carbon unit! 2 Acetyl-CoA  1 Oxaloacetate

Figure Glyoxylate Cycle and the Glyoxysome

Regulation of the Citric Acid Cycle

Regulatory Mechanisms Availability of substrates –Acetyl-CoA –Oxaloacetate –Oxygen (O 2 ) Need for citric acid cycle intermediates as biosynthetic precursors Demand for ATP

Table 17-2 Free Energy Changes of Citric Acid Cycle Enzymes

Regulation of Pyruvate Dehydrogenase Product Inhibition (competitive) –NADH –Acetyl-SCoA Phosphorylation/Dephosphorylation –PDH Kinase: inactivation –PDH Phosphatase: reactivation

Figure Covalent Modification and Regulation of PDH

Regulation of PDH Kinase (Inactivation) Activators –NADH –Acetyl-SCoA Inhibitors –Pyruvate –ADP –Ca 2+ (high Mg 2+ ) –K +

Regulation of PDH Phosphatase (Reactivation) Activators –Mg 2+ –Ca 2+

Regulation of Citrate Synthase Pacemaker Enzyme (rate-limiting step) Rate depends on availability of substrates –Acetyl-SCoA –Oxaloacetate

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

Regulation of  -Ketoglutarate Dehydrogenase Inhibitors –NADH –Succinyl-SCoA Activator: Ca 2+

Figure Regulation of the Citric Acid Cycle