Α-isopropylmalate Synthase Research Articles

Number and reactivity of the sulfhydryl groups of α-isopropylmalate synthase of Salmonella typhimurium

With three different methods of titration, 16 ± 1 free sulfhydryl groups were found per molecule of α-isopropylmalate synthase from Salmonella typhimurium, assuming a molecular weight of 160 000. On the basis of their reaction rate with 5,5′-dithio-bis-2-nitrobenzoate and with 2,2′-dithiodipyridine, at least two types of sulfhydryl groups could be distinguished. The presence of the endproduct inhibitor leucine or, to a lesser extent, of the substrate α-ketoisovalerate caused a general decrease of the sulfhydryl reactivity. Enzymatic activity was lost after 4–5 sulfhydryl groups had been titrated with 5,5′-dithio-bis-2-nitrobenzoate. Incubation with thiol compounds partially restored activity. The restoration was complicated by the finding that the thiol compounds themselves caused inactivation of the unmodified enzyme.

Dissociation of α-isopropylmalate synthase from [formula omitted] by its feedback inhibitor leucine

Results from sedimentation equilibrium experiments suggest that α-isopropylmalate synthase from Salmonella typhimurium exists in an association-dissociation equilibrium which is strongly influenced by its feedback inhibitor leucine. It is shown that under appropriate conditions leucine can cause a dissociation of the enzyme into units having the size of the polypeptide chains, which appear to be similar and to have a molecular weight of 45,000 – 50,000. The size of the oligomer has not been unambiguously established, although the data available favor the existence of a tetramer under conditions where the association-dissociation equilibrium is shifted toward the aggregated form of the enzyme.

α-Isopropylmalate Synthase from Salmonella typhimurium: PURIFICATION AND PROPERTIES

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)).