Isocitric acid binding

Gawron et al. (13,14) determined the stereochemistry of natural isocitric acid by chemical means. The results require the rrons-addition of water across the cis-aconitate intermediate double bond to produce either citrate or 2R,3S-isodtrate. Mass and NMR analyses of isotopically labeled citrate and isocitrate in the early 1960 s (15-17), defined the stercospedficities of the dehydration steps. These results led Gawron to propose the binding of cis-aconitate to the active site in two orientations differing by a 180° rotation about the double bond, as shown in Equation 2. This allows for the protonation by a base (-BH) and hydroxylation of the double bond to occur on aconitase at single, separate loci for the formation of either citrate or isocitrate. 

Phosphorylation of an enzyme can affect catalysis in another way by altering substrate-binding affinity. For example, when isocitrate dehydrogenase (an enzyme of the citric acid cycle Chapter 16) is phospho-rylated, electrostatic repulsion by the phosphoryl group inhibits the binding of citrate (a tricarboxylic acid) at the active site. 

Fluoroacetic acid has been identifled as the toxic component of the South African plant gijblaar (Dichapetalum cymosum) [34]. Its mechanism of action is based on inhibition of the citric acid cycle, the main source of metabolic energy in all animals [35]. In this cyde, fluoroacetate can replace acetate as a substrate of aconi-tase, an enzyme complex which usually forms dtrate by addition of acetate to a-oxoglutarate. The resulting fluorocitrate is binds tightly to the enzyme, but cannot be further converted to ds-aconitate and isocitrate [36], thus inhibiting aconitase. 

Rapid p-oxidation of fatty acids in perfused liver (DeBeer et a/., 1974) and in isolated mitochondria (Lopes-Cardozo and Van den Bergh, 1972) has been shown to suppress the operation of citric acid cycle apparently from the elevation of mitochondrial NADH/NAD ratio which restricts oxaloaceta-te availability for citrate synthase and simultaneously inhibits isocitrate oxidation (Lenartowicz et a/., 1976). Considerable support for an earlier postulate that oxaloacetate availability normally determines the rate of citrate synthesis has become available. Thus, because of marked protein binding, the concentration of free, as opposed to total, oxaloacetate in matrix of liver mitochondria is now estimated to be near the of citrate synthase (Siess et al., 1976 Brocks eta ., 1980). The antiketogenic effect of alanine (Nosadini et a/., 1980) and of 3-mercaptopicolinate, an inhibitor of phosphoenolpy-ruvate carboxykinase (Blackshear et a/., 1975), is believed to be exerted, at least in part, from their ability to raise hepatic oxaloacetate concentration. And, in pyruvate carboxylase deficiency, expected to impair oxaloacetate supply, concentration of ketone bodies is elevated (Saudubray et a/., 1976). 

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