Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) isozyme III from Escherichia coli has been studied in steady-state kinetic experiments in which the rates of formation of acetolactate (AL) and acetohydroxybutyrate (AHB) have been determined simultaneously. The ratio between the rates of production of the two alternative products and the concentrations of the substrates pyruvate and 2-ketobutyrate (2KB) leading to them, R, VAHB/VAL = R[( 2KB]/[pyruvate]), was found to be 40 +/- 3 under a wide variety of conditions. Because pyruvate is a common substrate in the reactions leading to both products and competes with 2-ketobutyrate to determine whether AL or AHB is formed, steady-state kinetic studies are unusually informative for this enzyme. At a given pyruvate concentration, the sum of the rates of formation of AL and AHB was nearly independent of the 2-ketobutyrate concentration. On the basis of these results, a mechanism is proposed for the enzyme that involves irreversible and rate-determining reaction of pyruvate, at a site which accepts 2-ketobutyrate poorly, if at all, to form an intermediate common to all the reactions. In the second phase of the reaction, various 2-keto acids can compete for this intermediate to form the respective acetohydroxy acids. 2-Keto acids other than the natural substrates pyruvate and 2-ketobutyrate may also compete, to a greater or lesser extent, in the second phase of the reaction to yield alternative products, e.g., 2-ketovalerate is preferred by about 2.5-fold over pyruvate. However, the presence of an additional keto acid does not affect the relative specificity of the enzyme for pyruvate and 2-ketobutyrate; this further supports the proposed mechanism. The substrate specificity in the second phase is an intrinsic property of the enzyme, unaffected by pH or feedback inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetohydroxy acid synthase (AHAS; EC 4.1.3.18) catalyzes the following two parallel, physiologically inportant reactions: condensation of two molecules of pyruvate to form acetolactate (AL), in the pathway to valine and leucine, and condensation of pyruvate plus 2-ketobutyrate to form acetohydroxybutyrate (AHB), in the pathway to isoleucine. We have determined the specificity ratio R with regard to these two reactions (where VAHB and VAL are rates of formation of the respective products) as follows: VMSH[2-ketobutyratel = R VAL lpyruvatel for 14 enzymes from 10 procaryotic and eucaryotic organisms. Each organism considered has at least one AHAS of R > 20, and some appear to contain but a single biosynthetic AHAS. The implications of this for the design of the pathway are discussed. The selective pressure for high specificity for 2-ketobutyrate versus pyruvate implies that the 2-ketobutyrate concentration is much lower than the pyruvate concentration in all these organisms. It seems important for 2-ketobutyrate levels to be relatively low to avoid a variety of metabolic interferences. These results also reinforce the conclusion that biosynthetic AHAS isozymes of low R (1 to 2) are a special adaptation for heterotrophic growth on certain poor carbon sources. Two catabolic "pH 6 AL-synthesizing enzymes" are shown to be highly specific for AL formation only (R < 0.1).In the pathway for the biosynthesis of branched-chain amino acids, the same set of enzymes catalyzes four consecutive reactions leading to two different sets of products, valine and leucine, on the one hand, and isoleucine on the other (40, 41) ( Fig. 1). Acetohydroxy acid synthase (AHAS, also known as acetolactate synthase; EC 4.1.3.18) catalyzes the first of these reactions, the irreversible decarboxylation of pyruvate and its condensation with either pyruvate or 2-ketobutyrate (15). The relative amounts of the two possible products formed by AHAS thus determines the relative amounts of valine plus leucine and isoleucine synthesized in these pathways.We have shown for a number of AHASs (3, 15) that the ratio of the relative rates of formation, V, of acetohydroxybutyrate (AHB) and acetolactate (AL) by a given enzyme is proportional to the ratio of the concentrations of the substrates 2-ketobutyrate and pyruvate and to a specificity ratio, R, characteristic of the enzyme VAHB VAL [2-ketobutyrate] = R [pyruvate] (1) Such a fixed specificity is expected if the competition between 2-ketobutyrate and pyruvate occurs on the enzyme subsequent to the irreversible and rate-determining formation of an intermediate from the first pyruvate (15).Pyruvate is a major intermediate in metabolism, whereas 2-ketobutyrate is a minor one, serving mainly as a precursor to isoleucine. Furthermore, 2-ketobutyrate may be toxic at high intracellular concentrations (7,24). In Escherichia coli grown on glucose, the concentration of pyruvate is nearly 2 orders of magnitude higher than that of 2-ketobutyrate (7,25). In order to synthesize similar quantities of each of the branch...
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