Kinetics and Mechanism of Acetohydroxy Acid Synthase Isozyme III from Escherichia coli

Natan Gollop, Batsheva Damri, Ze’ev Barak, David M. Chipman

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69 Scopus citations


Acetohydroxy acid synthase (AHAS, EC 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. The model for the enzyme that is suggested by the data is one in which the site for the competing second substrate has a specific and rather rigid nonpolar cavity of appropriate size to bind the CH3CH2 group of 2-ketobutyrate. Two other isozymes of AHAS from E. coli have also been studied and follow the same reaction mechanism. While AHAS II is similar to AHAS III in its substrate specificities, AHAS I has a much lower preference for 2-ketobutyrate (R = 2) and reacts very little with larger keto acids.

Original languageEnglish
Pages (from-to)6310-6317
Number of pages8
Issue number15
StatePublished - 1 Jan 1989

ASJC Scopus subject areas

  • Biochemistry


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