title: Mechanisms of catalysis and inhibition operative in the arginine deiminase from the human pathogen Giardia lamblia.
abstract: Giardia lamblia arginine deiminase (GlAD), the topic of this paper, belongs to the hydrolase branch of the guanidine-modifying enzyme superfamily, whose members employ Cys-mediated nucleophilic catalysis to promote deimination of l-arginine and its naturally occurring derivatives. G. lamblia is the causative agent in the human disease giardiasis. The results of RNAi/antisense RNA gene-silencing studies reported herein indicate that GlAD is essential for G. lamblia trophozoite survival and thus, a potential target for the development of therapeutic agents for the treatment of giardiasis. The homodimeric recombinant protein was prepared in Escherichia coli for in-depth biochemical characterization. The 2-domain GlAD monomer consists of a N-terminal domain that shares an active site structure (depicted by an insilico model) and kinetic properties (determined by steady-state and transient state kinetic analysis) with its bacterial AD counterparts, and a C-terminal domain of unknown fold and function. GlAD was found to be active over a wide pH range and to accept l-arginine, l-arginine ethyl ester, N(alpha)-benzoyl-l-arginine, and N(omega)-amino-l-arginine as substrates but not agmatine, l-homoarginine, N(alpha)-benzoyl-l-arginine ethyl ester or a variety of arginine-containing peptides. The intermediacy of a Cys424-alkylthiouronium ion covalent enzyme adduct was demonstrated and the rate constants for formation (k(1)=80s(-1)) and hydrolysis (k(2)=35s(-1)) of the intermediate were determined. The comparatively lower value of the steady-state rate constant (k(cat)=2.6s(-1)), suggests that a step following citrulline formation is rate-limiting. Inhibition of GlAD using Cys directed agents was briefly explored. S-Nitroso-l-homocysteine was shown to be an active site directed, irreversible inhibitor whereas N(omega)-cyano-l-arginine did not inhibit GlAD but instead proved to be an active site directed, irreversible inhibitor of the Bacillus cereus AD.
authors.affiliation: Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, NM 87131, USA.
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Li
authors.forename: Liudmila
authors.initials: L
authors.lastname: Kulakova
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.forename: Andrey
authors.initials: A
authors.lastname: Galkin
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Zhao
authors.forename: Theodore E
authors.initials: TE
authors.lastname: Nash
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Mariano
authors.forename: Osnat
authors.initials: O
authors.lastname: Herzberg
authors.forename: Debra
authors.initials: D
authors.lastname: Dunaway-Mariano
mesh_terms: D000818:Animals; D055162:Biocatalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D020868:Gene Silencing; D016829:Giardia lamblia / Q000201:enzymology*; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular
chemical_list: D006867:Hydrolases; C019723:arginine deiminase