Transition States and Control of Substrate Preference in the Promiscuous Phosphatase PP1

摘要

Catalytically promiscuous enzymes are an attractive frontier for biochemistry, because enzyme promiscuities not only plausibly explain enzyme evolution through the mechanism of gene duplication but also could provide an efficient route to changing the catalytic function of proteins by mimicking this evolutionary process. PPly is an effectively promiscuous phosphatase for the hydrolysis of both monoanionic and dianionic phosphate ester-based substrates. In addition to its native phosphate monoester substrate, PPl gamma catalyzes the hydrolysis of aryl methylphosphonates, fluorophosphate esters, phosphorothioate esters, and phosphodiesters, with second order rate accelerations that fall within the narrow range of 10(11)-10(13). In contrast to the different transition states in the uncatalyzed hydrolysis reactions of these substrates, PPl gamma catalyzes their hydrolysis through similar transition states. PPl gamma does not catalyze the hydrolysis of a sulfate ester, which is unexpected. The PPl gamma active site is tolerant of variations in the geometry of bound ligands, which permit the effective catalysis even of substrates whose steric requirements may result in perturbations to the positioning of the transferring group, both in the initial enzyme substrate complex and in the transition state. The conservative mutation of arginine 221 to lysine results in a mutant that is a more effective catalyst toward monoanionic substrates. The surprising conversion of substrate preference lends support to the notion that mutations following gene duplication can result in an altered enzyme with different catalytic capabilities and preferences and may provide a pathway for the evolution of new enzymes.