title: Contribution of an aspartate residue, D114, in the active site of clostridial glutamate dehydrogenase to the enzyme's unusual pH dependence.
abstract: Glutamate dehydrogenase from Clostridium symbiosum displays unusual kinetic behaviour at high pH when compared with other members of this enzyme family. Structural and sequence comparisons with GDHs from other organisms have indicated that the Asp residue at position 114 in the clostridial enzyme may account for these differences. By replacing this residue by Asn, a mutant protein has been created with altered functional properties at high pH. This mutant protein can be efficiently overexpressed in Escherichia coli, and several criteria, including mobility in non-denaturing electrophoresis, circular 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-type enzyme, and both CD data and activity measurements show that the distinctive time-dependent reversible conformational inactivation seen at high pH in the wild-type enzyme is abolished in the mutant.
authors.affiliation: Department of Biochemistry and Conway institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Ireland.
authors.forename: S
authors.initials: S
authors.lastname: Coughlan
authors.forename: X G
authors.initials: XG
authors.lastname: Wang
authors.forename: K L
authors.initials: KL
authors.lastname: Britton
authors.forename: T J
authors.initials: TJ
authors.lastname: Stillman
authors.forename: D W
authors.initials: DW
authors.lastname: Rice
authors.forename: R
authors.initials: R
authors.lastname: Chiaraluce
authors.forename: V
authors.initials: V
authors.lastname: Consalvi
authors.forename: R
authors.initials: R
authors.lastname: Scandurra
authors.forename: P C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D001224:Aspartic Acid / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003013:Clostridium / Q000201:enzymology*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
chemical_list: D017931:DNA Primers; D001224:Aspartic Acid; D005969:Glutamate Dehydrogenase