Human Paraoxonase I: A Potential Bioscavenger of Organophosphorus Nerve Agents

Human serum paraoxonase (HuPON1, EC 3.1.8.1) is a Ca2+-dependent enzyme that hydrolyzes esters including organophosphorus (OP) nerve agents. Efforts to elucidate the putative roles of active site amino acid residues have been hampered by the lack of three-dimensional structural information of this enzyme. The advent of an homology model for HuPON1 (folded onto the six-fold β-propeller structure of squid diisopropylfluorophosphatase) followed by a confirmatory crystal structure of a closely related hybrid PON molecule has led to the design and expression of site-directed mutants of HuPON1. These mutants were analyzed for enzymatic activity against a variety of substrates, including OP nerve agents. Substitution of residues predicted from the model to be important for substrate binding, Ca2+ ion coordination, and catalysis resulted in enzyme inactivation, supporting the validity of the proposed structural model. Mutants with altered specificities for substrate were identified; some recognized OP substrates as competitive inhibitors. The OP nerve agent soman (GD) has two chiral centers; the four stereoisomers of GD vary in toxicity by several orders of magnitude. A novel GC/MS-based assay was developed to examine the stereospecificity of wild-type and mutant HuPON1 enzymes for GD. The wild-type HuPON1 catalyzed hydrolysis of all four GD isomers with modest stereoselectivity for the less toxic (C±P+) isomers. Two of the mutants tested (S193A and S193G) demonstrated altered stereospecificity and kinetics, resulting in three to four-fold increased rate of hydrolysis for one of the toxic P- GD isomers. The capacity of recombinant HuPON1 to hydrolyze structurally isomeric OP nerve agents (VX and VR) was examined and found to differ in both affinity and rate of catalysis. Unlike VX, VR was not hydrolyzed by the H115W mutant of HuPON1, but instead was recognized as a competitive inhibitor. Given the structural similarity between VX and VR, these results suggest that residue H115 in wild-type HuPON1 is critical for determining the substrate specificity of HuPON1 for some classes of OPs. Together, the results presented have expanded our understanding of the amino acid residues critical for HuPON1 OP hydrolase activity. This information is being used to design mutants of PON1 with enhanced anti-OP activity and stereoselectivity towards the more toxic OP stereoisomers