The Effects of Loop Mutations on the Activity and Regulation of Malate Dehydrogenase

Abstract

Malate Dehydrogenase catalyzes the reversible reduction of Oxaloacetate using NADH, a reaction that plays a critical role in both cytosolic and mitochondrial metabolism. The reaction is regulated by both substrate inhibition and by citrate both of which appear to involve subunit communication in the homodimeric enzyme. To investigate the role that various turn regions in the molecule play in both catalysis and regulation we have, based upon clustal analysis, designed a variety of mutations to probe the role of specific residues in three specific turn regions, two near the subunit interface [residues 91‐95 and residues 203‐209‐ which appear to interact across the subunit interface] and one near the active site [residues 124‐129]. The mutations are expressed using a pQE vector and the proteins purified using Ni‐NTA resin. Initial rate kinetic studies show that various mutants affect Vmax, Km(NADH), Km(Oxaloacetate) or the substrate inhibition observed at high oxaloacetate concentrations as well as the ability of citrate to regulate the activity. The results further implicate subunit interactions as a critical component of the activity and regulation of the enzyme.Supported by NSF Grant MCB 0448905 to EB