1. Mechanisms of catalysis and inhibition operative in the arginine deiminase from the human pathogen Giardia lamblia
- Author
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Li, Zhimin, Kulakova, Liudmila, Li, Ling, Galkin, Andrey, Zhao, Zhiming, Knipp, Markus, Nash, Theodore E., Mariano, Patrick S., Herzberg, Osnat, and Dunaway-Mariano, Debra
- Subjects
Models, Molecular ,Tris ,Hydrolases ,Stereochemistry ,medicine.disease_cause ,Biochemistry ,Article ,chemistry.chemical_compound ,Drug Discovery ,Hydrolase ,Escherichia coli ,medicine ,Animals ,Giardia lamblia ,Gene Silencing ,Molecular Biology ,Arginine deiminase ,chemistry.chemical_classification ,biology ,Organic Chemistry ,Active site ,Kinetics ,Enzyme ,chemistry ,Biocatalysis ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Agmatine - 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.
- Published
- 2009