Modification and characterization of the neurotoxic substrate specificity of organophosphorous hydrolase

Abstract

Organophosphorus hydrolase (OPH) from P seudom onas diminuta MG is capable o f hydrolyzin g various organophosphorus com pounds. The structure-function relationship o f OPH was investigated utilizing a variety o f substrates with diverse side chains and leaving groups. The results demonstrated that the side chains greatly affected the catalytic efficiency o f OPH. The selected organophosphorothioates were hydrolyzed by OPH at varied rates, indicated by reaction with 5,5'-dithio-bis-nitrobenzoic acid. The hydrolytic reaction, inhibition o f acetylcholinesterase activity and paraoxon hydrolysis were further characterized with demeton-S. The roles o f potential active site residues, histidyl and cysteinyl residues, were investigated by site-directed mutagenesis. The mutant opd genes containing His to Asn and Cys to Ser replacement(s) were incorporated into expression vector pO P419-X , which constitutively expressed o pd gene product. The kinetics and the metal contents o f purified and the reconstituted enzym es were determined. The results are consistent with the hypothesis that the histidines at position 55, 57 and 201 are coordinated to a single metal ion, providing a metal center (M a ) for the formation o f the active site; H is230 is either directly involved in catalysis or is crucial for the stabilization of catalytic machinery; H is254 and His257 are potential ligands for the second metal ion (M b ). The kinetic and metal content analyses o f mutant enzym es with substitution at residues 254 and 257 by seven amino acid residues established the coordination o f H is254 and H is257 to the metal ion M b - Results supported the hypothesis that Mb bonded and oriented substrate when paraoxon, methyl parathion, nitrophenyl o-pinacolyl m ethyl phosphonate, diisopropyl fluorophosphoridate and dem eton-S were used as substrates. In the absence o f M b , the side chains o f residues 254 and 257 were proposed to interact with different alkyl side chains and probably with the leaving group o f substrate. Mutants with greatly increased soman and moderately improved VX activities, respectively, have been produced.