Probing the Subunit Interface of Malate Dehydrogenase: Effects of S266A and L269A Mutations

Abstract

Malate Dehydrogenase is thought to involve a reciprocating subunit mechanism involving ligand induced interactions across the subunit interface in the dimer and such a mechanism is thought to be involved in both catalysis and citrate regulation of this important enzyme. Clustal analysis of organelle forms of the enzyme and HINT analysis of interactions across the interface have identified two residues, S266 and L269 that could be involved in communication between the active site and the subunit interface. Two mutants, S266A and L269A have been constructed, expressed and purified by Nickel‐NTA affinity chromatography. Initial rate kinetic studies of each mutant reveal unique non‐ Michaelis‐Menten behavior. S266A shows linear Michaelis menten plots with Oxaloacetate as varied substrate but normal saturation with NADH while L269A shows non‐Michaelis Menten behavior with NADH as varied substrate but normal saturation with Oxaloacetate. To construct a heterodimer with one subunit containing the mutant while the other subunit is normal at the interface but unable to effectively bind Oxaloacetate as a result of mutation of upto three of the active site arginines, we have explored reversible dissociation of each relevant homo dimer to create heterodimers. Preliminary results suggest that while heterodimers can be separated by ion exchange chromatography the dynamic nature of the dimer formation creates problems. To overcome this complication we are exploring chemical cross‐linking of the dimer form of the enzyme with bifunctional imidates.