Roles of Hs205, His296, His303 and Asp259 in catalysis by NAD+-specific D-lactate dehydrogenase

The role of three histidine residues (His205, His296 and His303) and Asp259, important for the catalysis of NAD(+)-specific D-lactate dehydrogenase, was investigated using site-directed mutagenesis. None of these residues is presumed to be involved in coenzyme binding because K-m for NADH remained essentially unchanged for all the mutant enzymes. Replacement of His205 with lysine resulted in a 125-fold reduction in k(cat) and a slight lowering of the K-m value for pyruvate. D259N mutant showed a 56-fold reduction in k(cat) and a fivefold lowering of K-m. The enzymatic activity profile shifted towards acidic pH by approximate to 2 units. The H303K mutation produced no significant change in k(cat) values, although K-m for pyruvate increased fourfold. Substitution of His296 with lysine produced no significant change in k(cat) values or in K-m for substrate. The results obtained suggest that His205 and Asp259 play an important role in catalysis, whereas His303 does not. This corroborates structural information available for some members of the d-specific dehydrogenases family. The catalytic His296, proposed from structural studies to be the active site acid/base catalyst, is not invariant. Its function can be accomplished by lysine and this has significant implications for the enzymatic mechanism.