Tryptophan residue(s) as major components of the human serum paraoxonase active site.
Serum paraoxonase (PON1, EC 3.1.8.1.) is a high density lipid- (HDL)-associated, calcium-dependent enzyme whose 3D structure, active site residues and physiological substrates are not known. The kinetic parameters k(cat) and Km (relative to k(cat) and Km of the wild-type), determined with four substrates (phenylacetate, paraoxon, diazoxon and chlorpyrifosoxon) were less than 1, and more than 100% for the W280A and W280F mutant enzymes, respectively. These results indicated that the aromatic/hydrophobic character of the amino acid in position 280 is essential for PON1 activity. In this study, we investigated whether this aromatic residue is in the PON1 active site. Group-specific labelling studies with N-bromosuccinimide, an oxidative agent of tryptophan, strongly suggested that one or several Trp could be in the active site of PON1 but we could not conclude either on the specificity of the labelling reaction or on the number of oxidized Trp. However, although PON activity was not altered by the hydrophilic tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl chloride (NBC), it was significantly reduced by the p-nitrophenylacetate analog 2-acetoxy-5-nitrobenzyl chloride (ANBC), whose hydrolysis by PON1 generated NBC in the active site. Moreover, since at least one calcium ion is present in the PON catalytic site, we attempted to probe the metal local environment using the calcium analog terbium. The luminescence spectrum of the PON terbium complex exhibited an emission peak at 545 nm characteristic of an aromatic residue (Trp and/or Tyr)-terbium interaction. In conclusion, both the results obtained with the mechanism-based inhibitor of PON1 (ANBC) and the calcium-binding site luminescent probe terbium support the hypothesis of the presence of at least one Trp residue in the PON1 active site. Trp residue(s) may be involved in the binding of aromatic substrates.
Eppley Institute, University of Nebraska Medical Center, Omaha 68198-6805, USA. djosse@mail.unmc.edu
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D000085:Acetates / Q000494:pharmacology; D043303:Aryldialkylphosphatase; D001665:Binding Sites; D004390:Chlorpyrifos / Q000031:analogs & derivatives / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004950:Esterases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000737:chemistry / Q000235:genetics; D005470:Fluorometry; D006801:Humans; D007306:Insecticides / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009578:Nitrobenzenes / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000378:metabolism; D010648:Phenylacetates / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D013725:Terbium / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000085:Acetates; D004791:Enzyme Inhibitors; D007306:Insecticides; D009578:Nitrobenzenes; D009943:Organophosphorus Compounds; D010648:Phenylacetates; D013725:Terbium; D010649:Phenylalanine; C009618:O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphate; D014364:Tryptophan; C120619:2-acetoxy-5-nitrobenzyl chloride; C000912:diazoxon; D004950:Esterases; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; C025136:phenylacetic acid; D004390:Chlorpyrifos