Substrate binding is required for assembly of the active conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 A crystal structure of the glutaminase domain of glucosamine 6-phosphate synthase.

BACKGROUND: Amidotransferases use the amide nitrogen of glutamine in a number of important biosynthetic reactions. They are composed of a glutaminase domain, which catalyzes the hydrolysis of glutamine to glutamate and ammonia, and a synthetase domain, catalyzing amination of the substrate. To gain insight into the mechanism of nitrogen transfer, we examined the structure of the glutaminase domain of glucosamine 6-phosphate synthase (GLMS).
RESULTS: The crystal structures of the enzyme complexed with glutamate and with a competitive inhibitor, Glu-hydroxamate, have been determined to 1.8 A resolution. The protein fold has structural homology to other members of the superfamily of N-terminal nucleophile (Ntn) hydrolases, being a sandwich of antiparallel beta sheets surrounded by two layers of alpha helices.
CONCLUSIONS: The structural homology between the glutaminase domain of GLMS and that of PRPP amidotransferase (the only other Ntn amidotransferase whose structure is known) indicates that they may have diverged from a common ancestor. Cys1 is the catalytic nucleophile in GLMS, and the nucleophilic character of its thiol group appears to be increased through general base activation by its own alpha-amino group. Cys1 can adopt two conformations, one active and one inactive; glutamine binding locks the residue in a predetermined conformation. We propose that when a nitrogen acceptor is present Cys1 is kept in the active conformation, explaining the phenomenon of substrate-induced activation of the enzyme, and that Arg26 is central in this coupling.