Inactivation of the amidotransferase activity of carbamoyl phosphate synthetase by the antibiotic acivicin.
Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from 2 mol of ATP, bicarbonate, and glutamine. CPS was inactivated by the glutamine analog, acivicin. In the presence of ATP and bicarbonate the second-order rate constant for the inactivation of the glutamine-dependent activities was 4.0 x 10(4) m(-1) s(-1). In the absence of ATP and bicarbonate the second-order rate constant for inactivation of CPS was reduced by a factor of 200. The enzyme was protected against inactivation by the inclusion of glutamine in the reaction mixture. The ammonia-dependent activities were unaffected by the incubation of CPS with acivicin. These results are consistent with the covalent labeling of the glutamine-binding site located within the small amidotransferase subunit. The binding of ATP and bicarbonate to the large subunit of CPS must also induce a conformational change within the amidotransferase domain of the small subunit that enhances the nucleophilic character of the thiol group required for glutamine hydrolysis. The acivicin-inhibited enzyme was crystallized, and the three-dimensional structure was determined by x-ray diffraction techniques. The thiol group of Cys-269 was covalently attached to the dihydroisoxazole ring of acivicin with the displacement of a chloride ion.
Miles
Bryant W
BW
Department of Chemistry, Texas A & M University, College Station, Texas 77842-3012, USA.
Thoden
James B
JB
Holden
Hazel M
HM
Raushel
Frank M
FM
D000878:Anthranilate Synthase; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D007555:Isoxazoles / Q000494:pharmacology*; D007700:Kinetics; D008958:Models, Molecular; D019884:Nitrogenous Group Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D007555:Isoxazoles; D019884:Nitrogenous Group Transferases; D000878:Anthranilate Synthase; C020655:anthranilate synthase, glutamine amidotransferase subunit; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C006303:acivicin