D-2-HYDROXY-4-METHYLVALERATE DEHYDROGENASE FROM LACTOBACILLUS-DELBRUECKII SUBSP BULGARICUS .2. MUTAGENIC ANALYSIS OF CATALYTICALLY IMPORTANT RESIDUES

Five residues involved in catalysis and coenzyme binding have been ide ntified in D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacill us delbrueckii subsp. bulgaricus by using biochemical and genetical me thods. Enzyme inactivation with diethylpyrocarbonate indicated that a single histidine residue was involved in catalysis. Since H296 is the only conserved histidine in the whole family of NAD-dependent D-2-hydr oxyacid dehydrogenases, we constructed the H296Q and H296S mutants and showed that their catalytic efficiencies were reduced 10(5)-fold comp ared with the wild-type enzyme. This low residual activity was shown t o be insensitive to diethylpyrocarbonate. Taken together these data de monstrate that H296 is responsible for proton exchange in the redox re action. Two acidic residues (D259 and E264) were candidates for mainta ining H296 in the protonated state and their roles were examined by mu tagenesis. The D259N and E264Q mutant enzymes both showed similar and large reductions in their k(cat)/K-m ratios (200-800-fold, depending o n pH), indicating that either D259 or E264 (or both) could partner H29 6. The conserved R235 residue was a candidate for binding the alpha-ca rboxyl group of the substrate and it was changed to lysine. The R235K mutant showed a 104-fold reduced k(cat)/K-m due to both an increased K -m and a reduced k(cat) for 2-oxo-4-methylvalerate. Thus R235 plays a role in binding the substrate carboxylate similar to R171 in the L-lac tate dehydrogenases. Finally, we constructed the H205Q mutant to test the role of this partially conserved histidine residue (in 10/13 enzym es of the family). This mutant enzyme displayed a 7.7-fold increased k (cat) and a doubled catalytic efficiency at pH 5, was as sensitive to diethylpyrocarbonate as the wildtype but showed a sevenfold increased K-m for NADH and a 100-fold increase in K-d for NADH together with 10- 30-fold lower substrate inhibition. The transient kinetic behaviour of the H205Q mutant is as predicted from our previous study on the enzym atic mechanism of D-2-hydroxy-4-methylvalerate dehydrogenase which sho wed that coenzyme binding is highly pH dependent and indicated that re lease of the oxidised coenzyme is a significant component of the rate- limiting processes in catalysis at pH 6.5.