Stereospecificity of the Catalysis of GF by the Organophosphorus Acid Anhydrolase and Phosphotriesterse Enzymes

摘要

The stereospecificity of the catalysis of GF hydrolysis by several enzymes was investigated. Stereospecificity was not evident at higher (3 mM) GF concentrations but was clearly apparent at 0.5 mM GF. The differential effect was due to fluoride-catalyzed racemization of the substrate. The enzymes JD6.5 organophosphorus acid anhydrolase (OPAA), Alteromonas haloplanktis OPAA, and the wild-type phosphotriesterase (PTE) enzymes were all found to preferentially catalyze the hydrolysis of the (+)GF isomer, as determined by GC analysis of the remaining isomer. Acetylcholinesterase inhibition experiments showed the purified (-)GF isomer to be approximately twice as toxic as the racemic compound. One PTE mutant, H254G/H259W/L303T, was found to preferentially catalyze the hydrolysis of the (-)GF isomer, as shown by its complementation of JD6.5 OPAA and subsequent GC analysis of the remaining (+)GF isomer. Thus, either GF isomer could be individually prepared enzymatically and analyzed isothermally on a GC.