μ3-Oxo stabilized by three metal cations is a sufficient nucleophile for enzymatic hydrolysis of phosphate monoesters

Abstract

Diverse species have previously been proposed to be effective nucleophiles in the enzymatic hydrolysis of phosphate esters. A novel penta-metal cluster (two Fe³⁺ and three Ca²⁺) was recently discovered in the active site of PhoX alkaline phosphatase, with the revelation of the architecture of μ₃-oxo bridging one Ca²⁺ and two antiferromagnetically coupled Fe³⁺. In this work, using density functional theory calculations, the μ₃-oxo stabilized by three cations has been demonstrated to be a new type of effective nucleophile. The calculations give strong support to the “ping-pong” mechanism involving the nucleophilic attack of the μ₃-oxo on the substrate phosphor and the subsequent hydrolysis of the covalent phospho–enzyme intermediate. A base mechanism with the μ₃-oxo acting as a general base to activate an additional water molecule has further been demonstrated to be inaccessible. The results advance the understanding of the enzymatic hydrolysis of phosphate esters and may give important inspiration for the exploration of multinuclear biomimetic catalysts.