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)-associ ated, calcium-dependent enzyme whose 3D structure, active site residues and physiological substrates are not known. The kinetic parameters k(cat) and K-m (relative to k(cat) and K-m of the wild-type), determined with four sub strates (phenylacetate, paraoxon, diazoxon and chlorpyrifosoxon) were less than 1% and more than 100% for the W280A and W280F mutant enzymes, respecti vely. These results indicated that the aromatic/hydrophobic character of th e 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 agen t 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 reage nt 2-hydroxy-5-nitrobenzyl chloride (NBC), it was significantly reduced by the p-nitrophenylacetate analog 2-acetoxy-5-nitrobenzyl chloride (ANBC), wh ose 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 t o probe the metal local environment using the calcium analog terbium. The l uminescence spectrum of the PON-terbium complex exhibited an emission peak at 545 nm characteristic of an aromatic residue (Trp and/or Tyr)-terbium in teraction. In conclusion, both the results obtained with the mechanism-base d inhibitor of PON1 (ANBC) and the calcium-binding site luminescent probe t erbium support the hypothesis of the presence of at least one Trp residue i n the PON1 active site. Trp residue(s) may be involved in the binding of ar omatic substrates. (C) 1999 Elsevier Science Ireland Ltd. All rights reserv ed.