Reconsideration of the catalytic center and mechanism of mammalian paraoxonase/arylesterase.
For three decades, mammalian paraoxonase (A-esterase, aromatic esterase, arylesterase; PON, EC 3.1.8.1) has been thought to be a cysteine esterase demonstrating structural and mechanistic homologies with the serine esterases (cholinesterases and carboxyesterases). Human, mouse, and rabbit PONs each contain only three cysteine residues, and their positions within PON have been conserved. In purified human PON, residues Cys-41 and Cys-352 form an intramolecular disulfide bond and neither could function as an active-center cysteine. Highly purified, enzymatically 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 replaced with either serine (C283S) or alanine (C283A). The expressed C283 (wild-type) enzyme was inactivated by para-hydroxymercuribenzoate, but the C283S and C283A mutant enzymes were not inactivated. C283A and C283S mutant enzymes retained both paraoxonase and arylesterase activities, and the Km values for paraoxon and phenyl acetate were similar to those of the wild type. Clearly, residue Cys-283 is free in active PON, 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.
Sorenson
R C
RC
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0632, USA.
Primo-Parmo
S L
SL
Kuo
C L
CL
Adkins
S
S
Lockridge
O
O
La Du
B N
BN
D000595:Amino Acid Sequence; D000818:Animals; D043303:Aryldialkylphosphatase; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002384:Catalysis; D017124:Conserved Sequence; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D011994:Recombinant Proteins; D004950:Esterases; D043303:Aryldialkylphosphatase; D003545:Cysteine