Human paraoxonase double mutants hydrolyze V and G class organophosphorus nerve agents

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.