Kentaro Miyazaki

Kinetic analysis on the substrate specificity of 3-isopropylmalate dehydrogenase

Substrate specificity of 3‐isopropylmalate dehydrogenase is analyzed using a series of synthetic (2R,3S)‐3‐alkylmalates. Each analog with hydrogen, methyl, ethyl, isopropyl, isobutyl, tert‐butyl, and isoamyl group on C‐3 functions as a substrate, implying a broad substrate specificity of the enzyme toward alkylmalates. The incremental binding energy of the isopropyl group of 3‐isopropylmalate to the enzyme is estimated to be 3.55 kcal/mol, the rather small value supporting the broad specificity. Although the enzyme shows a broad specificity toward the alkylmalates, it does not show activity with isocitrate which has a negatively charged carboxymethyl group instead of the alkyl groups.

Tyr-139 in Thermus thermophilus 3-isopropylmalate dehydrogenase is involved in catalytic function

The role of Tyr‐139, which is thought to be located at the active site of Thermus thermophilus HB8 3‐isopropylmalate dehydrogenase, has been investigated by site‐specific replacement with phenylalanine. The replacement scarcely affected the Michaelis constant (K m) for 3‐isopropylmalate, but caused a 13‐fold decrease of that for NAD. The catalytic constant (k cat) showed a 14‐fold decrease. Accordingly, the catalytic efficiency (k cat/K m) decreased for 3‐isopropylmalate but not for NAD. The results suggest that Tyr‐139 is involved in the catalytic function through interaction with 3‐isopropylmalate.

Roles of Arg231 and Tyr284 of Thermus thermophilus isocitrate dehydrogenase in the coenzyme specificity

The coenzyme binding site of isocitrate dehydrogenase from Thermus thermophilus was analyzed by site‐directed mutagenesis. The mutation analysis revealed that Arg231 and Tyr284 are involved in the discrimination between NAD and NADP, suggesting that these two residues interact with 2′‐phosphate group of NADP.