Abstract
To get a closer insight into the substrate recognition of thermostable 3-isopropylmalate dehydrogenase (IPMDH) derived from Thermus thermophilus HB8, two analogs of the natural substrate, (2R,3S) 3-isopropylmalic acid (IPM) were synthesized according to the chiral transcription methodology which we have recently developed, and the kinetics of the enzyme reaction were analyzed. 2-O-methyl IPM was found to be inactive as a substrate but to function as an uncompetitive inhibitor, which suggests that, although it is the site of oxidation by NAD+, 2-O-methyl IPM was incorporated into one of the active sites of the homodimeric enzyme. The hydroxyl group at C-2 was not essential to the substrate recognition by IPMDH. The 1-carboxamide derivative of IPM was inactive, both as a substrate and as an inhibitor. This clearly implies the prime importance of electrostatic interaction between the C-1 carboxylate of IPM and a cationic function (probably a guanidino group of Arg-104) of the enzyme for the recognition of IPM.