1KEE

Inactivation of the Amidotransferase Activity of Carbamoyl Phosphate Synthetase by the Antibiotic Acivicin

Summary for 1KEE

• Entry DOI: 10.2210/pdb1kee/pdb
• Descriptor: Carbamoyl-phosphate synthetase large chain, Carbamoyl-phosphate synthetase small chain, MANGANESE (II) ION, ... (10 entities in total)
• Functional Keywords: atp grasp, channeling, antibiotic, ligase
• Biological source: Escherichia coli; More
• Total number of polymer chains: 8
• Total formula weight: 645960.47

Authors

Miles, B.W.,Thoden, J.B.,Holden, H.M.,Raushel, F.M. (deposition date: 2001-11-15, release date: 2001-12-21, Last modification date: 2023-08-16)

Primary citation

Miles, B.W.,Thoden, J.B.,Holden, H.M.,Raushel, F.M.
Inactivation of the amidotransferase activity of carbamoyl phosphate synthetase by the antibiotic acivicin.
J.Biol.Chem., 277:4368-4373, 2002

PubMed Abstract: 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.

PubMed: 11729189
DOI: 10.1074/jbc.M108582200

Experimental method

X-RAY DIFFRACTION (2.1 Å)