Contribution of an aspartate residue, D114, in the active site of clostridial glutamate dehydrogenase to the enzyme's unusual pH dependence

Glutamate dehydrogenase from Clostridium symbiosum displays unusual kinetic behaviour at high pH when compared with other members of this enzyme famil y. Structural and sequence comparisons with GDHs from other organisms have indicated that the Asp residue at position 114 in the clostridial enzyme ma y account for these differences. By replacing this residue by Asn, a mutant protein has been created with altered functional properties at high pH, Th is mutant protein can be efficiently overexpressed in Escherichia coli, and several criteria, including mobility in non-denaturing electrophoresis, ci rcular dichroism (CD) spectra and initial crystallisation studies, suggest a folding and an assembly comparable to those of the wild-type protein. The D114N mutant enzyme shows a higher optimum pH for activity than the wild-t ype enzyme, and both CD data and activity measurements show that the distin ctive time-dependent reversible conformational inactivation seen at high pH in the wild-type enzyme is abolished in the mutant. (C) 2001 Elsevier Scie nce B.V. All rights reserved.