Conformational flexibility in glutamate dehydrogenase. Role of water in substrate recognition and catalysis.
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.
Stillman
T J
TJ
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, U.K.
Baker
P J
PJ
Britton
K L
KL
Rice
D W
DW
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
D001426:Bacterial Proteins; D005971:Glutamates; D014867:Water; D009243:NAD; D005969:Glutamate Dehydrogenase