Catalytic Properties of Active Site Mutants of Glyoxasomal Malate Dehydrogenase

The active site of Malate Dehydrogenase contains a Histidine‐Aspartate pair where the aspartate is presumed to enhance the basicity of the catalytic histidine. In an attempt to more fully define the role of the various amino acid side chains in the active site cavity of Malate dehydrogenase, we have used site directed mutagenesis in conjunction with initial rate and stopped flow kinetics and structural determination. After the crystal structure was first determined by Banaszak and Bradshaw in the late 1970s, the focus of thought about the catalytic mechanism was on the His‐Asp pair in the active site cavity. The results presented here suggest a more complex role of many residues in the active site cavity in overall catalysis. The expressed proteins have been purified using NTA Chromatography and the purified proteins characterized using initial rate kinetics studies to give Km and Vmax values. Activation energies for the various mutants have been determined using temperature dependence of Vmax. The native enzyme exhibits significant substrate inhibition at high oxaloacetate concentrations. Stopped flow initial rate kinetics data demonstrates that catalysis is, in fact, the rate limiting step. The pH dependence of the various kinetic parameters suggests that the role of the histidine residue is more complex than simple base catalysis. The pH dependence data indicate that H182 might not be dominating the proton abstraction as the contemporary mechanism suggests; however, the greatly decreased turnover of the H182Q mutant does confirm H182 plays an integral role in catalysis. The crystal structure of the D157N mutant shows that in at least this mutant there is no significant change in structure This work is supported by NSF Grant MCB 0448905 to EB.