title: Conformational flexibility in glutamate dehydrogenase. Role of water in substrate recognition and catalysis.
abstract: We have solved the structure of the binary complex of the glutamate dehydrogenase from Clostridium symbiosum with glutamate to 1.9 A resolution. In this complex, the glutamate side-chain lies in a pocket on the enzyme surface and a key determinant of the enzymic specificity is an interaction of the substrate gamma-carboxyl group with the amino group of Lys89. In the apo-enzyme, Lys113 from the catalytic domain forms an important hydrogen bond to Asn373, in the NAD(+)-binding domain. On glutamate binding, the side-chain of this lysine undergoes a significant movement in order to optimize its hydrogen bonding to the alpha-carboxyl group of the substrate. Despite this shift, the interaction between Lys113 and Asn373 is maintained by a large-scale conformational change that closes the cleft between the two domains. Modelling studies indicate that in this "closed" conformation the C-4 of the nicotinamide ring and the alpha-carbon atom of the amino acid substrate are poised for efficient hydride transfer. Examination of the structure has led to a proposal for the catalytic activity of the enzyme, which involves Asp165 as a general base, and an enzyme-bound water molecule, hydrogen-bonded to an uncharged lysine residue, Lys125, as an attacking nucleophile in the reaction.
authors.affiliation: Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, U.K.
authors.forename: T J
authors.initials: TJ
authors.lastname: Stillman
authors.forename: P J
authors.initials: PJ
authors.lastname: Baker
authors.forename: K L
authors.initials: KL
authors.lastname: Britton
authors.forename: D W
authors.initials: DW
authors.lastname: Rice
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000648:ultrastructure; D001665:Binding Sites; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology; D018360:Crystallography, X-Ray; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000648:ultrastructure; D005971:Glutamates / Q000737:chemistry; D008958:Models, Molecular; D009038:Motion; D009243:NAD / Q000737:chemistry; D011487:Protein Conformation; D013379:Substrate Specificity; D014867:Water
chemical_list: D001426:Bacterial Proteins; D005971:Glutamates; D014867:Water; D009243:NAD; D005969:Glutamate Dehydrogenase