α-Isopropylmalate Synthase from Salmonella typhimurium

Abstract

Abstract α-Isopropylmalate synthase, the first enzyme in the biosynthesis of leucine, was purified to homogeneity from extracts of strain CV-19 of Salmonella typhimurium. From gel filtration and disc gel electrophoresis experiments it was deduced that the enzyme can exist in two physically distinguishable forms and that the equilibrium between these two forms is strongly and specifically influenced by leucine, the end product inhibitor. The pH optimum of the reaction was found to be at pH 8.5. The enzyme was about 30-fold more sensitive to leucine at pH 6.5 than at 8.5. Initial velocity studies revealed that the inhibition by leucine was noncompetitive with respect to α-ketoisovalerate and competitive with respect to acetyl coenzyme A. Under the conditions employed, there was no cooperation between α-ketoisovalerate binding sites. There was, however, increasing cooperation between at least two interacting acetyl coenzyme A binding sites in the presence of increasing concentrations of leucine. Cooperative interactions were also observed with leucine in the presence of substrates. Other inhibitors of the enzyme were α-ketoisocaproate, EDTA, and mercuric salts. The inhibition by leucine is discussed in terms of the model proposed by Monod, Wyman, and Changeux (J. Mol. Biol., 12, 88 (1965)).