THE ROLE OF GLUTAMATE-87 IN THE KINETIC MECHANISM OF THERMUS-THERMOPHILUS ISOPROPYLMALATE DEHYDROGENASE

The kinetic mechanism of the oxidative decarboxylation of 2R,3S-isopro pylmalate by the NAD-dependent isopropylmalate dehydrogenase of Thermu s thermaphilus was investigated. Initial rate results typical of rando m or steady-state ordered sequential mechanisms are obtained for both the wild-type and two mutant enzymes (E87G and E87Q) regardless of whe ther natural or alternative substrates (2R-malate, 2R,3S-tartrate and/ or NADP) are utilized. Initial rate data fail to converge on a rapid e quilibrium-ordered pattern despite marked reductions in specificity (k (cat)/K-m) caused by the mutations and alternative substrates. Althoug h the inhibition studies alone might suggest an ordered kinetic mechan ism with cofactor binding first, a detailed analysis reveals that the expected noncompetitive patterns appear uncompetitive because the diss ociation constants from the ternary complexes are far smaller than tho se from the binary complexes. Equilibrium fluorescence studies both co nfirm the random binding of substrates and the kinetic estimates of th e dissociation constants of the substrates from the binary complexes. The latter are not disturbed markedly by the mutations at site 87. Mut ations at site 87 do not affect the dissociation constants from the bi nary complexes, but do greatly increase the Michaelis constants, indic ating that E87 helps stabilize the Michaelis complex of the wild-type enzyme. The available structural data, the patterns of the kinetics re sults, and the structure of a pseudo-Michaelis complex of the homologo us isocitrate dehydrogenase of Escherichia col i suggest that E87 inte racts with the nicotinamide ring.