Purification and Properties of β-Isopropylmalate Dehydrogenase

Abstract β-Isopropylmalate dehydrogenase, an enzyme involved in leucine biosynthesis, was purified from crude extracts of nutritionally derepressed Salmonella typhimurium. Sedimentation of the enzyme in the ultracentrifuge results in a single, sharp, symmetrical schlieren peak. The s020,w is 5.0 S. Polyacrylamide gel electrophoresis at pH 9.4 and 7.5 showed single bands of protein. Sedimentation equilibrium studies with the native enzyme reveal a molecular weight of 70,000. Similar studies in 6 m guanidine hydrochloride-0.1 m β-mercaptoethanol yield a subunit molecular weight of 35,000. Tryptic peptide analysis suggests that the subunits have an identical amino acid sequence. Analysis of performic acid-oxidized enzyme shows 8 half-cystine residues per native enzyme molecule; spectrophotometric titration of the enzyme with p-hydroxymercuribenzoate indicate that no disulfide bonds are present. The Michaelis constant for β-isopropylmalate and nicotinamide adenine dinucleotide are 1.9 x 10-5 and 1.0 x 10-4 m, respectively. The enzyme activity is completely inhibited by p-hydroxymercuribenzoate and N-ethylmaleimide, but not by iodoacetate or iodoacetamide.