The Active Site Sulfenic Acid Ligand in Nitrile Hydratases Can Function as a Nucleophile

• Published in: Journal of the American Chemical Society Vol. 136; no. 4; pp. 1186 - 1189
• Main Authors: Martinez, Salette, Wu, Rui, Sanishvili, Ruslan, Liu, Dali, Holz, Richard
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 29.01.2014
• Subjects: Actinobacteria - enzymology; Biocatalysis; Boron Compounds - pharmacology; Boronic Acids - pharmacology; Catalysts; Catalytic Domain - drug effects; Communication; Enzyme Inhibitors - pharmacology; Enzymes; Hydration; Hydro-Lyases - antagonists & inhibitors; Hydro-Lyases - chemistry; Hydro-Lyases - metabolism; Inhibitors; Ligands; Models, Molecular; Nitriles; Nucleophiles; Structure-Activity Relationship; Sulfenic Acids - chemistry; Sulfenic Acids - metabolism; X-rays
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/ja410462j

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 (sp2) form to a tetrahedral form (sp3). 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 αCys113–OH sulfenic acid ligand. Based on these data, a new mechanism of action for the hydration of nitriles by NHase is presented.