Kinetic study of the side-door mutants of the model Carboxylesterase PNB CE
Streit, Timothy Michael
Carboxylesterases (CEs) metabolize a wide range of endogenous compounds and xenobiotics containing ester bonds. Crystal structures of mammalian CEs indicate a ?side door? located adjacent to the catalytic gorge that may act as an alternative pore for the trafficking of substrates and products. This study investigated the role of the ?gate? residue of the side door during para-Nitrobenzyl esterase (pnb CE)-catalyzed hydrolysis of esters. Purified recombinant pnb CE proteins were examined for their hydrolytic activity toward several esters. Mutation of the gate residue altered the kinetic parameters of pnb CE toward these substrates, demonstrated by increased Km values and decreased Vmax values. Site-specific mutations of the ?gate? residue also affected the sensitivity of the enzyme toward inhibiting organophosphate compounds. A distinct possibility is that the side door mutants affect substrate hydrolysis by increasing the steric hindrance and/or electrostatic repulsion between the substrate and the active site catalytic residues.