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

Abstract

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.