Enzyme-based detoxification of organophosphorus neurotoxic pesticides and chemical warfare agents

摘要

There are some 15,000 known organophosphorus chemicals. Some of these OP?s, including VX and paraoxon, demonstrate an acute neurotoxicity due to the inhibition of cholinergic enzymes. Organophosphorus chemical warfare agents and pesticide neurotoxins are subject to hydrolysis by OP degrading enzymes. To be useful as a bioremediation or anti-chemical warfare agent, the enzyme must be tailored for, and integrated into, a practical application platform. Several studies have established enzyme-based countermeasures, describing such diverse applications as decontaminating foams for surface remediation, encapsulating enzyme with liposome for in vivo therapy, enzyme attachments to surfaces for biosensors and development of a corn expression system for large-scale enzyme production. The goal of the research described here is to select, investigate and improve the operational potential of organophosphate-degrading enzymes including Organophosphorus Hydrolase (OPH, 3.1.8.1) and Organophosphorus Acid Anhydrolase (OPAA, 3.1.8.2). Using saturation kinetics, the catalytic efficiencies of these two major detoxification enzymes were characterized with substrates representing each class of OP neurotoxin, phosphotriester, phosphothioate and phosphofluoridate. OPH presents superior kinetic parameters with each OP class tested. Variants of OPH were created to increase the operational effectiveness of OP hydrolytic enzymes against phosphorothioates. An H254S/H257L mutation in the active site resulted in an improvement in the kinetics (kcat/KM) for the phosphorothioate, demeton-S. To screen potential vascular protection therapies, an in vitro protocol was developed to predict enzymatic effectiveness for protection of acetylcholinesterase from acute OP-inhibition. The protection abilities of the enzymes were directly related to their second order rate constants as inhibitory levels of OP are below the KM of the enzymes. Consideration of contaminant nature concentration and enzyme kinetic parameters, kcat and KM, is critical to understanding decontamination and effective use of enzyme technology. These technologies continue to develop and provide promising new decontamination tools for OP compounds.