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.

SUBMITTER: Li Z 

PROVIDER: S-EPMC4590290 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature