Elementary plant Biochemistry and Biotechnology (2+1)

The citric acid cycle

i.Formation of citrate

• The first step is the reaction between the four-carbon unit, oxaloacetate and the two-carbon unit, acetyl CoA resulting in the formation of citrate and coenzyme A catalysed by citrate synthase. The coenzyme A formed in this reaction is recycled.

ii. Formation of isocitrate via cis-aconitate

• The isomerization of citrate to isocitrate catalysed by aconitase occurs in two steps with the formation of cis-aconitate as an intermediate. This formation of isocitrate involves both dehydration and hydration. The result is an interchange of hydrogen and a hydroxyl group. In this reaction, fluoroacetate acts as an inhibitor to the enzyme, aconitase.

iii. Oxidation of isocitrate to α-ketoglutarate

• The enzyme, isocitrate dehydrogenase oxidatively decarboxylates isocitrate to α-ketoglutarate with simultaneous liberation of carbon dioxide. The intermediate in this reaction is oxalosuccinate, an unstable α-ketoacid. While bound to the enzyme, it loses carbon dioxide to form α-ketoglutarate. There are two different forms of isocitrate dehydrogenase (isozymes), one requiring NAD+ and other requiring NADP+.

iv. Oxidation of α-ketoglutarate to succinyl CoA

• α-Ketoglutarate, undergoes oxidative decarboxylation forming succinyl-CoA and carbon dioxide in the presence of α-ketoglutarate dehydrogenase complex, an assembly consisting of three kinds of enzymes. The mechanism of this reaction is very similar to the reaction catalyzed by pyruvate dehydrogenase complex. This reaction is irreversible. Arsenite acts as an inhibitor of TCA cycle by inhibiting the action of α-ketoglutarate dehydrogenase complex.

v. Conversion of succinyl CoA to succinate

• Succinate is formed in a reversible reaction from succinyl CoA catalysed by the enzyme, succinyl CoA synthetase or succinate thiokinase with the simultaneous formation of GTP and coenzyme A. Succinate thiokinase utilises GDP in animal tissues whereas it uses ADP predominantly in plants and bacteria. The formation of GTP in this reaction is a substrate level phosphorylation reaction.

vi. Formation of fumarate by oxidation of succinate

• The succinate formed from succinyl CoA is oxidised to fumarate by succinate dehydrogenase with the participation of FAD. Malonate, an analogue of succinate being a strong competitive inhibitor of succinate dehydrogenase, blocks the citric acid cycle.

vii. Formation of malate by hydration of fumarate

• The reversible hydration of fumarate to L-malate is catalysed by fumarase.

viii. Oxidation of malate to oxaloacetate

• This reaction forms the last reaction of the citric acid cycle. NAD-linked malate dehydrogenase catalyses the oxidation of L-malate to oxaloacetate.Energetics of tricarboxylic acid cycle. From one molecule of glucose, two molecules of pyruvate are formed which in turn give rise to two molecules of acetyl CoA. When two molecules of acetyl-CoA undergo oxidation through TCA cycle, the following number of high-energy bonds (ATPs) is produced.