Defining the Role of Active Site Residues in the Activity of Glyoxysomal Malate Dehydrogenase

Abstract

Although the structure of Malate Dehydrogenase has long been known and a mechanism proposed involving a his‐asp pair in the active site, little is known about the roles that residues further from the active site may play. Analysis of the active site cavity, both visually and by QM‐MM approaches, led to the construction of active site mutants and second sphere mutants [involving residues located 5–10A from the site of catalysis]. Using site directed mutagenesis, kinetics studies and x ray crystallography the roles of both residues directly in the active site and those further afield have been examined. H220 and D193 play well characterized roles in proton abstraction, however the pH dependence of the D193N mutant suggest other factors may be involved. Second sphere residues, N124, T152, T186, A183, G180, V164, N165, S166 and H90 have been mutated, expressed and studied by kinetics and stability studies. N165D has similar Vmax values at pH 7 & 8 but a marked increase in substrate inhibition at pH 7 and a reversal of the pH effects on Km relative to the native protein or S166A. The N165D and the S166A mutants have been crystallized and 3D structure determination is in progress. It is clear that a number of residues within this second sphere contribute to protein stability and aid in the fine tuning of the regulatory effects of substrate inhibition and pH Dependence.Supported by NF Grant MCB 0448905 to Ellis Bell