THE GLUTAMINE HYDROLYSIS FUNCTION OF HUMAN GMP SYNTHETASE - IDENTIFICATION OF AN ESSENTIAL ACTIVE-SITE CYSTEINE

GMP synthetase (EC 6.3.5.2) is an amidotransferase that catalyzes the amination of xanthosine 5'-monophosphate to form GMP in the presence o f glutamine and ATP, Glutamine hydrolysis produces the necessary amino group while ATP hydrolysis drives the reaction, Ammonia can also serv e as an amino group donor, GMP synthetase contains two functional doma ins, which are well coordinated, The ''glutamine amide transfer'' or g lutaminase domain is responsible for glutamine hydrolysis, The synthet ase domain is responsible for ATP hydrolysis and GMP formation. Inorga nic pyrophosphate inhibits the synthetase and uncouples the two domain functions by allowing glutamine hydrolysis to take place in the absen ce of ATP hydrolysis or GMP formation, Acivicin, a glutamine analog, s electively abolishes the glutaminase activity, It inhibits the synthet ase activity only when glutamine is the amino do nor, When ammonia is used in place of glutamine, acivicin has no effect on the synthetase a ctivity, Acivicin inhibits GRIP synthetase irreversibly by covalent mo dification. Enzyme inactivation is greatly facilitated by the presence of substrates. Acivicin labels GMP synthetase at a single site, and a tryptic peptide containing the modified residue was isolated, Mass sp ectrometry and Edman sequence analysis show that Cys(104) is the site of modification, This residue is conserved among GRIP synthetases and is located within a predicted glutamine amide transfer domain, These d ata suggest that Cys(104) is an essential residue involved in the hydr olysis of glutamine to produce an amino group and is not needed for th e hydrolysis of ATP or amination of xanthosine 5'-monophosphate to pro duce GMP.