Human serum paraoxonase (PON1): identification of essential amino acid residues by group-selective labelling and site-directed mutagenesis

Human serum paraoxonase/arylesterase (PON1, EC 3.1.8.1.) is a calcium-depen dent enzyme which hydrolyzes a wide variety of organophosphates, including paraoxon, DFP, sarin and soman. Although the 3-D structure of PON has not y et been determined and its sequence shows no similarity with any other crys tallized proteins, we undertook to identify some of its essential amino aci d residues by two complementary approaches: group-specific labelling and si te-directed mutagenesis. Group-specific labelling studies, performed on the purified native enzyme, indicated that one or more Trp, His and Asp/Glu ar e potentially important residues for PON activity. Based on these results, we identified some of these residues, conserved in the sequenced mammalian PON1, by site-directed mutagenesis. PON1 mutants were transiently expressed in 293T cells. The catalytic constants k(cat) and K-m (relative to k(cat) and K-m of the wild-type) determined with four different substrates (phenyl acetate, paraoxon, diazoxon, chlorpyrifos oxon), were not significantly cha nged for the following mutants: W193A, W201A, W253A, H160N, H245N, H250N, H 347N, E32A, E48A, D88A, D107A, D121A, D273A. By contrast, k(cat) was less t han 1% for eight mutants: W280A, H114N, H133N, H154N, H242N, H284N, E52A an d D53A. The essential amino acid residues identified in this work could be part of the PON1 active site, acting either as calcium ligands (E52 and D53 ?) or as' substrate binding (W280?) or nucleophilic (His residues?) sites. However, we cannot rule out that the effects of mutations on catalytic prop erties resulted from a remote conformational change and/or misfolding of mu tant proteins. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.