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Chiral Hydrolysis of G‐type Nerve Agents by Human Prolidase
Author(s) -
Kajih Takwen,
Cerasoli Douglas,
Harrison Joshua,
Norris Joseph,
Targiani Robert
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb124
Subject(s) - chemistry , in vivo , hydrolysis , enantiomer , nerve agent , in vitro , enzyme , stereochemistry , recombinant dna , gene isoform , biochemistry , biology , acetylcholinesterase , gene , microbiology and biotechnology
Human prolidase is a bioscavenger of organophosphorus nerve agents that catalyzes the hydrolysis of GA, GB, GD, and GF. Chiral gas chromatography/mass spectrometry was used to measure the rate of hydrolysis of racemic nerve agent, yielding a relative measure of the stereopreference of prolidase for the enantiomers of GA, GB, GD, and GF. Each of the nerve agents analyzed consists of P(+) and P(−) isomers, of which the P(−) isomer is more inhibitory of AChE in vitro and more toxic in vivo. Both skin and kidney isoforms of prolidase were expressed as recombinant proteins in insect larvae and purified (>95% by Chesapeake‐PERL); no functional differences between the wild‐type isoforms were detected. Catalytic efficiencies for the different G agents varied between 10 4 to 10 6 M −1 min −1 , and preference for the P(+) isomer was detected for each agent. Based on these results, wild‐type prolidase is not predicted to be capable of affording protection in vivo against G agents. We also attempted to enhance the OP hydrolyzing activity of prolidase by producing three variants: R395S, R398S and T299Y. None of the variants improved the catalytic efficiencies sufficiently to be predicted to afford protection. Future efforts to improve the hydrolytic activity of prolidase toward OP nerve agents will utilize the knowledge obtained here in the generation of a directed evolution library.