SUBSTRATE-BINDING IS REQUIRED FOR ASSEMBLY OF THE ACTIVE CONFORMATIONOF THE CATALYTIC SITE IN NTN AMIDOTRANSFERASES - EVIDENCE FROM THE 1.8 ANGSTROM 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 gl utaminase domain, which catalyzes the hydrolysis of glutamine to gluta mate and ammonia, and a synthetase domain, catalyzing amination of the substrate. To gain insight into the mechanism of nitrogen transfer, w e examined the structure of the glutaminase domain of glucosamine g-ph osphate synthase (GLMS), Results: The crystal structures of the enzyme complexed with glutamate and with a competitive inhibitor, Glu-hydrox amate, have been determined to 1.8 Angstrom resolution, The protein fo ld has structural homology to other members of the superfamily of N-te rminal nucleophile (Ntn) hydrolases, being a sandwich of antiparallel beta sheets surrounded by two layers of a helices, Conclusions: The st ructural homology between the glutaminase domain of GLMS and that of P RPP amidotransferase (the only other Ntn amidotransferase whose struct ure is known) indicates that they may have diverged from a common ance stor. Cys1 is the catalytic nucleophile in GLMS, and the nucleophilic character of its thiol group appears to be increased through general b ase activation by its own alpha-amino group. Cys1 can adopt two confor mations, one active and one inactive; glutamine binding locks the resi due in a predetermined conformation. We propose that when a nitrogen a cceptor is present Cys1 is kept in the active conformation, explaining the phenomenon of substrate-induced activation of the enzyme, and tha t Arg26 is central in this coupling.