Purification and Properties of the Acetohydroxy Acid Isomeroreductase of Salmonella typhimurium

Abstract

α-Acetohydroxy acid isomeroreductase, which catalyzes both the second step specific to valine biosynthesis and the third step specific to isoleucine biosynthesis, has been purified from derepressed Salmonella typhimurium. The enzyme appears to be homogeneous in the ultracentrifuge and by immunodiffusion in agar. The molecular weight, as determined by sedimentation equilibrium ultracentrifugation, is approximately 220,000. The enzyme is stereospecific for the B side of NADPH. At pH 7.5, the optimal pH for the isomeroreductase, the reversibility of the reaction cannot be shown, but at higher values the reaction is readily reversible. The pH optimum of the reverse reaction is about 9.4. The purified enzyme catalyzes the isomerization of α-keto-β-hydroxyisovalerate to α-acetolactate and the NADPH-linked reduction of α-keto-β-hydroxyisovalerate to α,β-dihydroxyisovalerate as well as the over-all conversion of α-acetolactate to α,β-dihydroxyisovalerate. The ratio of these activities remains constant during purification and the coenzyme and Mg++ requirements suggest that the isomerase and reductase activities are inherent properties of the enzyme. However, experiments with labeled substrate indicate that α-keto-β-hydroxyisovalerate is not a free intermediate of the over-all reaction.