RECONSIDERATION OF THE CATALYTIC CENTER AND MECHANISM OF MAMMALIAN PARAOXONASE ARYLESTERASE

For three decades, mammalian paraoxonase (A-esterase, aromatic esteras e, arylesterase; PON, EC 3.1.8.1) has been thought to be a cysteine es terase demonstrating structural and mechanistic homologies with the se rine esterases (cholinesterases and carboxyesterases). Human, mouse, a nd rabbit PONs each contain only three cysteine residues, and their po sitions within PON have been conserved, In purified human PON, residue s Cys-41 and Cys-352 form an intramolecular disulfide bond and neither could function as an active-center cysteine, Highly purified, enzymat ically active PON contains a single titratable sulfhydryl group, Thus, Cys-283 is the only probable candidate for an active-center cysteine, Through site-directed mutagenesis of the human cDNA, Cys-283 was repl aced with either serine (C283S) or alanine (C283A), The expressed C283 (wild type) enzyme was inactivated by para-hydroxymercuribenzoate, bu t the C283S and C283A mutant enzymes were not inactivated, C283A and C 283S mutant enzymes retained both paraoxonase and arylesterase activit ies, and the K-m values for paraoxon and phenyl acetate were similar t o those of the wild type, Clearly, residue Cys-283 is free in active P ON, but a free sulfhydryl group is not required for either paraoxonase or arylesterase activities, Consequently, it is necessary to examine other models for the active-site structure and catalytic mechanism of PON.