Identification of residues essential for human paraoxonase (PON1) arylesterase/organophosphatase activities.
Human serum paraoxonase (PON1) is a calcium-dependent organophosphatase. To identify residues essential for PON1 activity, we adopted complementary approaches based on chemical modification and site-directed mutagenesis. To detect 45Ca2+ binding to native and chemically modified PON1, we performed nondenaturating gel electrophoresis. The environment of calcium-binding sites was probed using the Ca2+ analogue, terbium. Tb3+ binds to calcium-binding sites as shown by displacement of 45Ca2+ by Tb3+. Binding of Tb3+ is accompanied by a complete loss of enzyme activity. PON1 chemical modification with the Trp-selective reagent, N-bromosuccinimide, and the Asp/Glu-selective, dicyclohexylcarbodiimide, established that Trp and Asp/Glu residues are components of the PON1 active center and calcium-binding sites. Additional evidence for the presence of a Trp residue in the PON1 calcium-binding sites was a characteristic fluorescence emission at 545 nm from the PON1-Tb3+ complex and abolishment of that fluorescence upon modification by N-bromosuccinimide. The importance of aromatic/hydrophobic character of the residue 280 was demonstrated by site-directed mutagenesis: the W280F mutant was fully active while the W280A and W280L mutants had markedly reduced activity. Twelve amino acids among conserved His and Asp/Glu residues were found essential for PON1 arylesterase and organophosphatase activities: H114, H133, H154, H242, H284, D53, D168, D182, D268, D278, E52, and E194. Finally, the cysteines constituting the PON1 disulfide bond (C41 and C352) were essential, but the glycan chains linked to Asn 252 and 323 were not essential for PON1 secretion and activity.
Unité d'enzymologie, Centre de Recherches du Service de Santé des Armées, La Tronche, France. 101511.3606@compuserve.com
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D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D043303:Aryldialkylphosphatase; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D002132:Calcium Radioisotopes / Q000378:metabolism; D002460:Cell Line; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004950:Esterases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D006031:Glycosylation; D006639:Histidine / Q000235:genetics; D006801:Humans; D007202:Indicators and Reagents; D007668:Kidney; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013725:Terbium; D014364:Tryptophan / Q000235:genetics
D000596:Amino Acids; D002132:Calcium Radioisotopes; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D013725:Terbium; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D004024:Dicyclohexylcarbodiimide; D014364:Tryptophan; D004950:Esterases; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; D001974:Bromosuccinimide; D004047:Diethyl Pyrocarbonate