The Citrate Cycle

How do redox reactions in the Citrate Cycle contribute to energy conversion?

● The citrate cycle is the hub of metabolism for three reasons: (1) it is central to aerobic metabolism by generating the bulk of the NADH and FADH2 used to generate ATP by oxidative phosphorylation; (2) it oxidizes metabolic fuels from a variety of sources (carbohydrates, fatty acids, proteins); and (3) it provides metabolites for biosynthetic pathways.

● The citrate cycle accomplishes four things with each turn of the cycle: (1) it decarboxylates citrate to remove two carbons contributed by acetyl-CoA; (2) it transfers eight electrons to 3 NAD1 and 1 FAD; (3) it generates 1 GTP (converted to ATP by nucleoside diphosphate kinase) by substrate-level phosphorylation; and (4) it regenerates oxaloacetate so the cycle can start again.

● The citrate cycle is also called the Krebs cycle in honor of Hans Krebs, who discovered it; alternatively, it is also called the citric acid cycle or the tricarboxylic acid (TCA) cycle. The descriptive term citrate cycle reflects that all three carboxylate groups on citrate are deprotonated at physiologic pH – it is not an acid in cells at pH 7.2.

● The amount of energy available from a coupled redox reaction is directly related to the difference between two reduction potentials and is defined by the term delta E°’. The delta E°’ of a coupled redox reaction is determined by subtracting the E°’ of the reductant (e^– donor) from the E°’of the oxidant (e^– acceptor).

What is the mechanism by which the enzyme pyruvate dehydrogenase converts pyruvate to acetyl-CoA?

● Pyruvate is oxidatively decarboxylated in the mitochondrial matrix by the multi-subunit pyruvate dehydrogenase complex, which uses a five-step reaction mechanism that requires three distinct enzymes and five different coenzymes. The five coenzymes used by the pyruvate dehydrogenase complex are NAD⁺, FAD, CoA, TPP, and α-lipoic acid (lipoamide).

● Thiamine, also called vitamin B1, is the precursor to TPP, an important coenzyme in the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase reactions. Thiamine deficiency causes the human disease beriberi.

● The eukaryotic pyruvate dehydrogenase complex contains three enzymes: E1, pyruvate dehydrogenase; E2, dihydrolipoyl acetyltransferase; and E3, dihydrolipoyl dehydrogenase.

● Pyruvate dehydrogenase activity is regulated by allosteric control in response to energy charge, NADH to NAD⁺  ratios, and CoA to acetyl-CoA ratios. It is also regulated by serine phosphorylation, which is mediated by kinase and phosphatase enzymes that are themselves regulated by energy charge metabolites.

What are the eight enzymatic reactions of the Citrate Cycle?

● The citrate cycle consists of eight enzymatic reactions: four of these are coupled redox reactions that transfer electrons to NAD⁺ and FAD; one is a substrate-level phosphorylation reaction generating GTP (converted to ATP by nucleoside diphosphate kinase); and the other three reactions include two isomerizations and one hydration.

● Citrate synthase catalyzes the first reaction in the pathway, which is the condensation of oxaloacetate and acetyl-CoA to form citrate. Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate by transferring two electrons to NAD⁺ to form NADH, releasing CO2 in the process.

● α-Ketoglutarate dehydrogenase is functionally similar to pyruvate dehydrogenase and requires the same five coenzymes. It catalyzes an oxidative decarboxylation reaction that produces CO2, NADH, and succinyl-CoA.

● Cytosolic aconitase is a dual-function protein that generates isocitrate for various metabolic pathways. It also binds specific sequences in mRNAs encoding iron-metabolizing proteins.

● GTP (or ATP) is generated by a substrate-level phosphorylation reaction catalyzed by the enzyme succinyl-CoA synthetase. GTP is readily converted to ATP in cells by the enzyme nucleoside diphosphate kinase.

How is metabolic flux through the Citrate Cycle regulated?

● The three main control points for regulation of the citrate cycle are the reactions catalyzed by citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase.

● Regulation of citrate cycle reactions is accomplished by several mechanisms, including substrate availability, product inhibition, and feedback inhibition.

● Citrate synthase is inhibited by citrate, succinyl-CoA, NADH, and ATP; inhibition by ATP is reversed by ADP. Isocitrate dehydrogenase is activated by ADP and Ca²⁺ and inhibited by NADH and ATP. α-Ketoglutarate dehydrogenase is activated by Ca²⁺ and AMP and is inhibited by NADH, succinyl-CoA, and ATP.

What is the metabolic fate of the Citrate Cycle intermediates?

● The citrate cycle provides biosynthetic precursors for several metabolic pathways; it is considered to be an amphibolic pathway because it functions in both catabolism and anabolism.

● Excess citrate in mitochondria is exported to the cytosol, where it is cleaved by the enzyme citrate lyase to release acetyl-CoA and oxaloacetate. Cytosolic acetyl-CoA is used for fatty acid and cholesterol biosynthesis, whereas oxaloacetate is used to generate phosphoenolpyruvate for gluconeogenesis.

● Oxaloacetate and α-ketoglutarate are metabolic precursors to aspartate and glutamate, respectively, and succinyl-CoA is a precursor in heme biosynthesis. Malate can be used as a source of carbon in the gluconeogenic pathway.

● The enzyme pyruvate carboxylase balances the input of oxaloacetate and acetyl-CoA into the citrate cycle by converting pyruvate into oxaloacetate using an ATP-dependent carboxylation reaction that is stimulated by acetyl-CoA.

● The pyruvate carboxylase reaction is anaplerotic (meaning to “fill up”), because it replenishes citrate cycle intermediates by providing oxaloacetate. Phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase also supply oxaloacetate to the citrate cycle, and malic enzyme supplies malate.