title: Human paraoxonase double mutants hydrolyze V and G class organophosphorus nerve agents.
abstract: Variants of human paraoxonase 1 (PON1) are being developed as catalytic bioscavengers for the organophosphorus chemical warfare agents (OP). It is preferable that the new PON1 variants have broad spectrum hydrolase activities to hydrolyze both G- and V-class OPs. H115W PON1 has shown improvements over wild type PON1 in its capacity to hydrolyze some OP compounds. We improved upon these activities either by substituting a tryptophan (F347W) near the putative active site residues for enhanced substrate binding or by reducing a bulky group (Y71A) at the periphery of the putative enzyme active site. When compared to H115W alone, we found that H115W/Y71A and H115W/F347W maintained VX catalytic efficiency but showed mixed results for the capacity to hydrolyze paraoxon. Testing our double mutants against racemic sarin, we observed reduced values of K(M) for H115W/F347W that modestly improved catalytic efficiency over wild type and H115W. Contrary to previous reports, we show that H115W can hydrolyze soman, and the double mutant H115W/Y71A is nearly 4-fold more efficient than H115W for paraoxon hydrolysis. We also observed modest stereoselectivity for hydrolysis of the P(-) stereoisomer of tabun by H115W/F347W. These data demonstrate enhancements made in PON1 for the purpose of developing an improved catalytic bioscavenger to protect cholinesterase against chemical warfare agents.
authors.affiliation: US Army Medical Research Institute of Chemical Defense, Research Division, Physiology and Immunology Branch, 3100 Ricketts Point Road, Aberdeen Proving Grounds, MD 21010-5400, USA. stephen.drew.kirby@us.army.mil
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Kirby
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Norris
authors.forename: J
authors.initials: J
authors.lastname: Richard Smith
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Bahnson
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Cerasoli
mesh_terms: D019943:Amino Acid Substitution; D000931:Antidotes / Q000378:metabolism; D043303:Aryldialkylphosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D002619:Chemical Warfare Agents / Q000145:classification / Q000378:metabolism* / Q000633:toxicity; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism / Q000633:toxicity; D009943:Organophosphorus Compounds / Q000145:classification / Q000378:metabolism* / Q000633:toxicity; D009946:Organothiophosphorus Compounds / Q000378:metabolism / Q000633:toxicity; D010261:Paraoxon / Q000145:classification / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012524:Sarin / Q000378:metabolism / Q000633:toxicity; D012999:Soman / Q000378:metabolism / Q000633:toxicity; D013379:Substrate Specificity
chemical_list: D000931:Antidotes; D043303:Aryldialkylphosphatase; D002619:Chemical Warfare Agents; D050505:Mutant Proteins; D010755:Organophosphates; D009943:Organophosphorus Compounds; D009946:Organothiophosphorus Compounds; D010261:Paraoxon; D011994:Recombinant Proteins; D012524:Sarin; D012999:Soman; C009680:VX-agent; C468406:PON1 protein, human; C009374:tabun