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-dependent enzyme which hydrolyzes a wide variety of organophosphates, including paraoxon, DFP, sarin and soman. Although the 3-D structure of PON has not yet been determined and its sequence shows no similarity with any other crystallized proteins, we undertook to identify some of its essential amino acid residues by two complementary approaches: group-specific labelling and site-directed mutagenesis. Group-specific labelling studies, performed on the purified native enzyme, indicated that one or more Trp, His and Asp/Glu are 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 Km (relative to k(cat) and Km of the wild-type) determined with four different substrates (phenylacetate, paraoxon, diazoxon, chlorpyrifos oxon), were not significantly changed for the following mutants: W193A, W201A, W253A, H160N, H245N, H250N, H347N, E32A, E48A, D88A, D107A, D121A, D273A. By contrast, k(cat) was less than 1% for eight mutants: W280A, H114N, H133N, H154N, H242N, H284N, E52A and 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 properties resulted from a remote conformational change and/or misfolding of mutant proteins.
Josse
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Eppley Institute, University of Nebraska Medical Center, Omaha 68198-6805, USA. djosse@mail.unmc.edu
Xie
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Masson
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Lockridge
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D000601:Amino Acids, Essential / Q000097:blood* / Q000235:genetics* / Q000378:metabolism; D043303:Aryldialkylphosphatase; D001974:Bromosuccinimide / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004789:Enzyme Activation; D004950:Esterases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000601:Amino Acids, Essential; D004024:Dicyclohexylcarbodiimide; D004950:Esterases; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; D001974:Bromosuccinimide; D004047:Diethyl Pyrocarbonate