title: The active site sulfenic acid ligand in nitrile hydratases can function as a nucleophile.
abstract: Nitrile hydratase (NHase) catalyzes the hydration of nitriles to their corresponding commercially valuable amides at ambient temperatures and physiological pH. Several reaction mechanisms have been proposed for NHase enzymes; however, the source of the nucleophile remains a mystery. Boronic acids have been shown to be potent inhibitors of numerous hydrolytic enzymes due to the open shell of boron, which allows it to expand from a trigonal planar (sp(2)) form to a tetrahedral form (sp(3)). Therefore, we examined the inhibition of the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) by boronic acids via kinetics and X-ray crystallography. Both 1-butaneboronic acid (BuBA) and phenylboronic acid (PBA) function as potent competitive inhibitors of PtNHase. X-ray crystal structures for BuBA and PBA complexed to PtNHase were solved and refined at 1.5, 1.6, and 1.2 Å resolution. The resulting PtNHase-boronic acid complexes represent a "snapshot" of reaction intermediates and implicate the cysteine-sulfenic acid ligand as the catalytic nucleophile, a heretofore unknown role for the αCys(113)-OH sulfenic acid ligand. Based on these data, a new mechanism of action for the hydration of nitriles by NHase is presented.
authors.affiliation: Department of Chemistry, Marquette University , Milwaukee, Wisconsin 53201, United States.
authors.forename: Salette
authors.initials: S
authors.lastname: Martinez
authors.forename: Rui
authors.initials: R
authors.lastname: Wu
authors.forename: Ruslan
authors.initials: R
authors.lastname: Sanishvili
authors.forename: Dali
authors.initials: D
authors.lastname: Liu
authors.forename: Richard
authors.initials: R
authors.lastname: Holz
mesh_terms: D039903:Actinobacteria / Q000201:enzymology*; D055162:Biocatalysis; D001896:Boron Compounds / Q000494:pharmacology; D001897:Boronic Acids / Q000494:pharmacology; D020134:Catalytic Domain / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D013329:Structure-Activity Relationship; D013434:Sulfenic Acids / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001896:Boron Compounds; D001897:Boronic Acids; D004791:Enzyme Inhibitors; D008024:Ligands; D013434:Sulfenic Acids; C030433:n-butylboronic acid; D006836:Hydro-Lyases; C048831:nitrile hydratase; C010686:benzeneboronic acid