Analysis of active-site amino-acid residues of human serum paraoxonase using competitive substrates.
Serum paraoxonase (PON1) is a calcium-dependent six-fold beta-propeller protein structurally similar to the di-isopropylfluorophosphatase (DFPase) found in the squid Loligo vulgaris. Human serum paraoxonase (HuPON1) has been shown to hydrolyze an array of substrates even though relatively little is known about its physiological role(s) or its catalytic mechanism. Through site-directed mutagenesis studies, designed from a DFPase-like homology model, and from a crystal structure of a hybrid PON1 molecule, amino-acid residues essential for enzyme function, including H115 and F222, have been identified. It was shown previously that, when H115 is replaced with tryptophan, the resulting enzyme hydrolyzes paraoxon but not phenyl acetate. This study shows that, when present simultaneously, phenyl acetate competitively inhibits paraoxon hydrolysis by H115W. Conversely, when F222 is replaced with tyrosine, mutant F222Y can hydrolyze phenyl acetate but not paraoxon. The presence of DFP, an inhibitor of both arylesterase and paraoxonase activities of wild-type HuPON1 (mean Ki=0.48+/-0.15 mM), has no effect on the ability of F222Y to catalyze the hydrolysis of phenyl acetate, suggesting that the F222Y mutant is unable to bind DFP. Together, the results suggest that, in wild-type HuPON1, H115 and F222 are important in determining substrate binding and specificity, but are not likely to be directly involved in substrate hydrolysis.
Yeung
David T
DT
Biochemical Pharmacology Branch, Pharmacology Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400, USA.
Lenz
David E
DE
Cerasoli
Douglas M
DM
D000595:Amino Acid Sequence; D000818:Animals; D043303:Aryldialkylphosphatase / Q000037:antagonists & inhibitors / Q000097:blood / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010648:Phenylacetates / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
D010648:Phenylacetates; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D007531:Isoflurophate; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; C025136:phenylacetic acid