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TCA cycle Carmen Sato­Bigbee, Ph.D. Objectives: 1) To show the reactions involved in the TCA cycle. 2) To understand the function of the TCA cycle and its regulation in relation to other metabolic pathways and energy requirements of the cells. Resources: Lehninger et al, Principles of Biochemistry 2005 ; Marks et al., Medical Biochemistry, 2005. The tricarboxylic acid (TCA) cycle (citric acid cycle or Krebs cycle) occurs in mitochondria and is the final common pathway for the oxidation of fuel molecules, not only carbohydrates but also fatty acids and amino acids. Most fuel molecules enter the TCA cycle as acetyl coenzyme A (acetyl CoA). In this cycle acetyl CoA is oxidized to CO2. Electrons released in different steps of this pathway are transferred to NAD + or FAD producing 3 NADH and 1 FADH2 molecules per molecule of acetyl CoA. These molecules are then oxidized by O2 in the electron transport chain producing about 3 ATP per NADH and 2 ATP per FADH2. Figure 1­ Taken from Lehninger et al.
Figure 2­ Taken from Lehninger et al. Linkage between glycolysis and the TCA cycle: Pyruvate + CoA + NAD + → Acetyl CoA + CO2 + NADH Oxidative decarboxylation catalyzed by pyruvate dehydrogenase complex, requires thiamine pyrophosphate as cofactor. (thiamine= vitamin B1). Thiamine deficiency results in Beriberi, with neurological and cardiovascular problems. Patients show high levels of pyruvate in blood after glucose ingestion.
Figure 3­ Taken from Lehninger et al. (Principles of Biochemistry, 2000) This reaction is: inhibited by Acetyl CoA, NADH, and ATP. activated by pyruvate, NAD + and CoA.
Steps of the TCA cycle: Figure 4­ Taken from Lehninger et al.
Step 1: Condensation of Oxaloacetate with Acetyl CoA to produce Citrate oxaloacetate + acetyl CoA + H2O → citrate + CoA + H + Catalyzed by citrate synthase. Hydrolysis of Acetyl CoA (a high energy compound) is an exothermic process, )G 0 ’= ­7.7 kcal. Step 2: Isomerization of citrate into isocitrate citrate ↔ cis­aconitate ↔ isocitrate Catalyzed by aconitase. Involves a dehydration and a hydration step with cis­aconitate as intermediate. )G 0 ’= +1.5 kcal Step 3: Isocitrate oxidation and decarboxylation to "­ketoglutarate isocitrate + NAD + → "­ketoglutarate + NADH + H + + CO2 Catalyzed by isocitrate dehydrogenase. Involves oxidation of alcohol group in isocitrate to a ketone intermediate, oxalosuccinate. Decarboxylation of oxalosuccinate generates "­ketoglutarate. )G 0 ’= ­ 5.3 kcal Step 4: Oxidative decarboxylation of "­ketoglutarate results in formation of succinyl CoA "­ketoglutarate + NAD + + CoA → succinyl CoA + NADH + CO2 Catalyzed by "­ketoglutarate dehydrogenase (like pyruvate dehydrogenase, requires thiamine pyrophosphate. Low enzyme activity in Beriberi). )G 0 ’= ­ 8 kcal. Step 5: Conversion of succinyl CoA into succinate with formation of a high energy phosphate compound succinyl CoA + Pi + GDP ↔ succinate + GTP + CoA Catalyzed by succinyl CoA synthase. Cleavage of succinyl CoA releases enough energy to generate GTP from GDP. Only step in the TCA cycle that directly generates a high­energy phosphate compound. )G 0 ’= ­ 0.7 kcal. GTP can be used to regenerate ATP in a reaction catalyzed by nucleoside diphosphate kinase= GTP + ADP ↔ GDP + ATP. Steps 6, 7, and 8: Regeneration of oxaloacetate by oxidation of succinate
6) succinate + FAD ↔ fumarate + FADH2 Catalyzed by succinate dehydrogenase, enzyme directly linked to the electron transport chain. )G 0 ’= 0. Uses FAD because the free energy change is not enough to generate NADH. 7) fumarate + H2O ↔ malate Catalyzed by fumarase. )G 0 ’= 0. 8) malate + NAD + ↔ oxaloacetate + NADH Catalyzed by malate dehydrogenase. )G 0 ’= +7.1 kcal. Although )G 0 ’ is positive, under physiological conditions is thermodynamically reversible. Oxaloacetate concentration is maintained low because is used in the first step of the cycle. Net reaction: Acetyl CoA + 3 NAD + + FAD + GDP + Pi + 2 H20 → 2 CO2 + CoA +3 NADH+ 3 H + FADH2 + GTP Regulation: As with other pathways involved in producing energy, the TCA cycle will be regulated according to the energy levels of the cells. Too much ATP and NADH (that will finally result in more ATP) will be inhibitors. ADP, which signals use of ATP, will be activator. [Regulation and function go together]. Pyruvate dehydrogenase (connection with glycolysis): Inhibited by acetyl CoA, NADH, and ATP. Activated by pyruvate, NAD + and CoA. Citrate synthase (step 1): Inhibited by citrate. Isocitrate dehydrogenase (step 3): activated by ADP and Ca 2+ . Inhibited by NADH. "­Ketoglutarate dehydrogenase (step 4): Activated by Ca 2+ . Inhibited by NADH and succinyl CoA. Malate dehydrogenase (step 8): Too much NADH inhibits the regeneration of oxaloacetate.
Anaplerotic or “filling up” reactions: The function of these reactions is to compensate for the efflux of intermediates from the TCA cycle into other pathways. The carbon skeleton of some amino acids comes from intermediates of the TCA cycle. In nervous tissue "­Ketoglutarate is transformed to glutamate and GABA, both neurotransmitters. During fasting, malate can be used to make glucose by gluconeogenesis. In liver, citrate is converted to acetyl CoA and used in fatty acid synthesis. Succinyl CoA is used in heme synthesis. Anaplerotic reactions include: ­ Pyruvate carboxylase reaction: Alternate way of using pyruvate to make oxaloacetate. Important in liver and nervous tissue because they have a constant efflux of intermediates from the TCA cycle. ­ Conversion of amino acids (isoleucine, valine, methionine) and odd chain fatty acids into succinyl CoA. ­ Conversion of amino acids to fumarate and oxaloacetate. ­ Conversion of amino acids to "­Ketoglutarate. Review questions 1) What is the function of the TCA cycle? 2) What are the steps of the TCA cycle? 3) What is produced in the TCA cycle? In what steps? What is the fate of those molecules? 4)Are there any steps that produce ATP? Directly? Indirectly? 5) How do different fuel molecules enter in the TCA cycle? 6) At what steps is the TCA cycle regulated? By what molecules? 7) How does the regulation of the TCA cycle relate to glycolysis, ATP levels in the cells, O2 levels?