title: Arginine deiminase uses an active-site cysteine in nucleophilic catalysis of L-arginine hydrolysis.
abstract: Arginine deiminase (EC 3.5.3.6) catalyzes the hydrolysis of l-arginine to citrulline and ammonium ion, which is the first step of the microbial l-arginine degradation pathway. The deiminase conserves the active-site Cys-His-Asp motif found in several related enzymes that catalyze group-transfer reactions from the guanidinium center of arginine-containing substrates. For each of these enzymes, nucleophilic catalysis by the conserved Cys has been postulated but never tested. In this communication we report the results from rapid quench studies of single-turnover reactions carried out with recombinant Pseudomonas aeruginosa arginine deiminase and limiting [14C-1]l-arginine. The citrulline-formation and arginine-decay curves measured at 25 degrees C were fitted to yield apparent rate constants k = 3.6 +/- 0.1 s-1 and k = 4.2 +/- 0.1 s-1, respectively. The time course for the formation (k =13 s-1) and decay (k = 6.5 s-1) of 14C-labeled enzyme defined a kinetically competent intermediate. Under the same reaction conditions, the Cys406Ser mutant failed to form the 14C-labeled enzyme intermediate. These results, along with the recently reported enzyme X-ray structure (Galkin, A.; Kulakova, L.; Sarikaya, E.; Lim, K.; Howard, A.; Herzberg, O. J. Biol. Chem. 2004, 279, 14001-14008, evidence a reaction pathway in which l-arginine deimination proceeds via a covalent enzyme intermediate formed by ammonia displacement from the arginine guanidinum carbon by the active-site Cys406.
authors.affiliation: Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, USA.
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Lu
authors.forename: Andrey
authors.initials: A
authors.lastname: Galkin
authors.forename: Osnat
authors.initials: O
authors.lastname: Herzberg
authors.forename: Debra
authors.initials: D
authors.lastname: Dunaway-Mariano
mesh_terms: D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D015394:Molecular Structure; D009954:Ornithine Carbamoyltransferase / Q000378:metabolism; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa; D013237:Stereoisomerism
chemical_list: D001120:Arginine; D009954:Ornithine Carbamoyltransferase; D017851:Phosphotransferases (Carboxyl Group Acceptor); C019288:carbamate kinase; D006867:Hydrolases; C019723:arginine deiminase; D003545:Cysteine