KINETIC MECHANISM AND REACTION PATHWAY OF THERMUS-THERMOPHILUS ISOPROPYLMALATE DEHYDROGENASE

Mechanistic studies of isopropylmalate dehydrogenase (IMDH, EC 1.1.1.85),the penultimate enzyme in leucine biosynthesis in bacteria and yeast, have been conducted. It performs two chemical operations, oxidation and decarboxylation, on isopropylmalate to produce alpha-ketoisocaproate. A recombinant enzyme encoded by the leuB gene of the thermophilic bacterium T. thermophilus was used for experiments addressing the kinetic mechanism and chemical pathway of IMDH. A new, asymmetric synthesis of the substrate, (2R,3S)-isopropylmalate, has been developed starting from (2R,3R)tartaric acid. On the basis of kinetic inhibition patterns and exchange experiments, it has been shown that the enzyme follows an ordered sequential bi-tri mechanism, with cofactor NAD binding before substrate beta-isopropylmalate. The release of products occurs in the order CO2, alpha-ketoisocaproate, and NADH. The enzyme catalyzes the exchange of solvent protons into the cu-position of the product, implying that an enol/enolate intermediate is formed. The enzyme conducts two discrete steps: dehydrogenation to isopropyloxaloacetate, which was prepared and shown to be a competent substrate, and decarboxylation to give product.