Isocitrate, aconitase reaction oxidation

Aconitase is an iron-sulfur protein, or nonheme iron protein. It contains four iron atoms that are not incorporated as part of a heme group. The four iron atoms are complexed to four inorganic sulfides and three cysteine sulfur atoms, leaving one iron atom available to bind citrate and then isocitrate through their carboxylate and hydroxyl groups (Figure 17,12). This iron center, in conjunction with other groups on the enzyme, facilitates the dehydration and rehydration reactions. We will consider the role of these iron-sulfur clusters in the electron-transfer reactions of oxidative phosphorylation subsequently (Section 18.3.1). 

Citrate, a tertiary alcohol, is next converted into its isomer, isocitrate, a secondary alcohol. The isomerization occurs in two steps, both of which are catalyzed by the same aconitase enzyme. The initial step is an E2 dehydration of a /3-hydroxy acid, the same sort of reaction that occurs in step 9 of glycolysis (Figure 29.4). The second step is a conjugate nucleophilic addition of water to the C=C bond, the same sort of reaction that occurs in step 2 of the /3 oxidation pathway (Figure 29.2). 

The second metabolic pathway which we have chosen to describe is the tricarboxylic acid cycle, often referred to as the Krebs cycle. This represents the biochemical hub of intermediary metabolism, not only in the oxidative catabolism of carbohydrates, lipids, and amino acids in aerobic eukaryotes and prokaryotes, but also as a source of numerous biosynthetic precursors. Pyruvate, formed in the cytosol by glycolysis, is transported into the matrix of the mitochondria where it is converted to acetyl CoA by the multi-enzyme complex, pyruvate dehydrogenase. Acetyl CoA is also produced by the mitochondrial S-oxidation of fatty acids and by the oxidative metabolism of a number of amino acids. The first reaction of the cycle (Figure 5.12) involves the condensation of acetyl Co and oxaloacetate to form citrate (1), a Claisen ester condensation. Citrate is then converted to the more easily oxidised secondary alcohol, isocitrate (2), by the iron-sulfur centre of the enzyme aconitase (described in Chapter 13). This reaction involves successive dehydration of citrate, producing enzyme-bound cis-aconitate, followed by rehydration, to give isocitrate. In this reaction, the enzyme distinguishes between the two external carboxyl groups... 

Citrate is isomerized to form a secondary alcohol that can be easily oxidized. In the next reaction of the cycle, citrate, which contains a tertiary alcohol, is reversibly converted to isocitrate by aconitase. During this isomerization reaction, an intermediate called cw-aconitate is formed by dehydration. The carbon-carbon double bond of cw-aconitate is then rehydrated to form the more reactive secondary alcohol, isocitrate. 

In the next step of the TCA cycle, the hydroxyl (alcohol) group of citrate is moved to an adjacent carbon so that it can be oxidized to form a keto group. The isomerization of citrate to isocitrate is catalyzed by the enzyme aconitase, which is named for an intermediate of the reaction. The enzyme isocitrate dehydrogenase catalyzes the oxidation of the alcohol group and the subsequent cleavage of the carboxyl group to release CO2 (an oxidative decarboxylation). 

Various relationships between enzymes of the Krebs cycle and mitochondria are possible. For instance, all enzymes could be enclosed within mitochondrial structures or the enzymes could take part in the structural build-up of the cell. There is no evidence demonstrating that all enzymes of the Krebs cycle are part of the mitochondria. The existence of enzymes with multiple catalytic properties (isocitric dehydrogenase, aconitase, and malic dehydrogenase) and the failure to separate the multiple steps of an overall reaction (pyruvic and a-ketoglutarate oxidation) are sometimes taken as evidence for the participation of the enzyme in the building-up of the mitochondrial structure, but these arguments do not take into account the limitations of the actual biochemical methods, and, therefore, conclusions based upon them are premature. 

The citric acid cycle is the series of enzyme-catalyzed reactions responsible for the oxidation of the acetyl group of acetyl-CoA to two molecules of CO2. The enzymes that catalyze the reactions are 1. citrate synthase 2. aconitase 3. isocitrate dehydrogenase 4. a-ketoglutarate dehydrogenase 5. succinyl-CoA synthetase 6. succinate dehydrogenase 7. fumarase and 8. malate dehydrogenase. 

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