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 of glutamine and ATP. Glutamine hydrolysis produces the necessary amino group while ATP hydrolysis drives the reaction. Ammonia can also serve as an amino group donor. GMP synthetase contains two functional domains, which are well coordinated. The "glutamine amide transfer" or glutaminase domain is responsible for glutamine hydrolysis. The synthetase domain is responsible for ATP hydrolysis and GMP formation. Inorganic pyrophosphate inhibits the synthetase and uncouples the two domain functions by allowing glutamine hydrolysis to take place in the absence of ATP hydrolysis or GMP formation. Acivicin, a glutamine analog, selectively abolishes the glutaminase activity. It inhibits the synthetase activity only when glutamine is the amino donor. When ammonia is used in place of glutamine, acivicin has no effect on the synthetase activity. Acivicin inhibits GMP synthetase irreversibly by covalent modification. 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 spectrometry and Edman sequence analysis show that Cys104 is the site of modification. This residue is conserved among GMP synthetases and is located within a predicted glutamine amide transfer domain. These data suggest that Cys104 is an essential residue involved in the hydrolysis of glutamine to produce an amino group and is not needed for the hydrolysis of ATP or amination of xanthosine 5'-monophosphate to produce GMP.
Nakamura
J
J
Institute of Biochemistry and Cell Biology, Syntex Discovery Research, Palo Alto, California 94303, USA.
Straub
K
K
Wu
J
J
Lou
L
L
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases; D003545:Cysteine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005973:Glutamine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D006157:Guanosine Monophosphate / Q000096:biosynthesis; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007555:Isoxazoles / Q000494:pharmacology; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification
D004791:Enzyme Inhibitors; D007555:Isoxazoles; D010446:Peptide Fragments; D005973:Glutamine; D006157:Guanosine Monophosphate; D000255:Adenosine Triphosphate; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing); D003545:Cysteine; C006303:acivicin