ABSTRACT
The recent use of organophosphate nerve agents in Syria, Malaysia, Russia, and the United Kingdom has reinforced the potential threat of their intentional release. These agents act through their ability to inhibit human acetylcholinesterase (hAChE; E.C. 3.1.1.7), an enzyme vital for survival. The toxicity of hAChE inhibition via G-series nerve agents has been demonstrated to vary widely depending on the G-agent used. To gain insight into this issue, the structures of hAChE inhibited by tabun, sarin, cyclosarin, soman, and GP were obtained along with the inhibition kinetics for these agents. Through this information, the role of hAChE active site plasticity in agent selectivity is revealed. With reports indicating that the efficacy of reactivators can vary based on the nerve agent inhibiting hAChE, human recombinatorially expressed hAChE was utilized to define these variations for HI-6 among various G-agents. To identify the structural underpinnings of this phenomenon, the structures of tabun, sarin, and soman-inhibited hAChE in complex with HI-6 were determined. This revealed how the presence of G-agent adducts impacts reactivator access and placement within the active site. These insights will contribute toward a path of next-generation reactivators and an improved understanding of the innate issues with the current reactivators.
Subject(s)
Acetylcholinesterase/metabolism , Cholinesterase Inhibitors/adverse effects , Nerve Agents/adverse effects , Oximes/adverse effects , Pyridinium Compounds/adverse effects , Acetylcholinesterase/chemistry , Acetylcholinesterase/isolation & purification , Cholinesterase Inhibitors/chemistry , Humans , Molecular Structure , Nerve Agents/chemistry , Oximes/chemistry , Pyridinium Compounds/chemistryABSTRACT
The bacterial enzyme phosphotriesterase (PTE) is noted for its ability to hydrolyze many organophosphate compounds, including insecticides and chemical warfare agents. PTE has been the subject of multiple enzyme evolution attempts, which have been highly successful against specific insecticides and the G-type nerve agents. Similar attempts targeting the V-type nerve agents have failed to achieve the same degree of success. Enzyme evolution is an inherently complex problem, which is complicated by synergistic effects, the need to use analogues in high-throughput screening, and a lack of quantitative data to direct future efforts. Previous evolution experiments with PTE have assumed an absence of synergy and minimally screened large libraries, which provides no quantitative information about the effects of individual mutations. Here a systemic approach has been applied to a 28800-member six-site PTE library. The library is screened against multiple V-agent analogues, and a combination of sequence and quantitative activity analysis is used to extract data about the effects of individual mutations. We demonstrate that synergistic relationships dominate the evolutionary landscape of PTE and that analogue activity profiles can be used to identify variants with high activity for substrates. Using these approaches, multiple variants with kcat/ Km values for the hydrolysis of VX that were improved >1500-fold were identified, including one variant that is improved 9200-fold relative to wild-type PTE and is specific for the SP enantiomer of VX. Multiple variants that were highly active for ( SP)-VR were identified, the best of which has a kcat/ Km values that is improved 13400-fold relative to that of wild-type PTE.
Subject(s)
Bacterial Proteins/chemistry , Chemical Warfare Agents/chemistry , Organophosphorus Compounds/chemistry , Organothiophosphorus Compounds/chemistry , Phosphoric Triester Hydrolases/chemistry , Adaptation, Physiological/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chemical Warfare Agents/metabolism , Decontamination , Directed Molecular Evolution , Hydrolysis , Mutation , Organophosphates/chemistry , Organophosphates/metabolism , Organophosphorus Compounds/metabolism , Organothiophosphorus Compounds/metabolism , Phosphoric Triester Hydrolases/genetics , Phosphoric Triester Hydrolases/metabolism , Pseudomonas/enzymology , Pseudomonas/genetics , Stereoisomerism , Substrate SpecificityABSTRACT
Ultra-Performance Liquid Chromatography/electrospray ionization mass spectrometry was used for the trace level determination of isopropyl methylphosphonofluoridate (Sarin, GB) and ( O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX) after extraction from various foods. A method utilizing normal phase silica gel was developed for the sample preparation and extraction of VX and GB from food. The extraction efficiencies of the normal phase silica gel method for VX was compared to those of other commercial solid phase extraction media and was found to be comparable. Sarin was found to be incompatible with both the mixed mode cation exchange (MCX) sorbents and QuEChERS methods that are commercially available but was successful with the normal phase silica gel method. The linear range of quantitation for VX was 0.1-330 ng/mL and for GB was 20-1200 ng/mL. The average recoveries of VX and GB from the various food matrices along with the corresponding relative standard deviations (RSDs) are reported.
Subject(s)
Chemical Warfare Agents/chemistry , Chemical Warfare Agents/isolation & purification , Chromatography, High Pressure Liquid/methods , Food Contamination/analysis , Organothiophosphorus Compounds/analysis , Organothiophosphorus Compounds/isolation & purification , Sarin/analysis , Sarin/isolation & purification , Solid Phase Extraction/methods , Tandem Mass Spectrometry/methods , Animals , Eggs/analysis , Fruit and Vegetable Juices/analysis , Milk/chemistry , Silica Gel , Solid Phase Extraction/instrumentationABSTRACT
The wild-type OPAA enzyme has relatively high levels of catalytic activity against several organophosphate G-type nerve agents. A series of mutants containing replacement amino acids at the OPAA Y212, V342, and I215 sites showed several fold enhanced catalytic efficiency on sarin, soman, and GP. One mutant, Y212F/V342L, showed enhanced stereospecificity on sarin and that enzyme along with a phosphotriesterase mutant, GWT, which had the opposite stereospecificity, were used to generate enriched preparations of each sarin enantiomer. Inhibition of acetylcholinesterase by the respective enantioenriched sarin solutions subsequently provided identification of the sarin enantiomers as separated by normal phase enantioselective liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometry.
Subject(s)
Aryldialkylphosphatase/genetics , Aryldialkylphosphatase/metabolism , Nerve Agents/metabolism , Amino Acid Sequence , Amino Acid Substitution , Biocatalysis , Kinetics , Mutagenesis, Site-Directed , Organophosphorus Compounds/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sarin/metabolism , Soman/metabolism , Stereoisomerism , Substrate SpecificityABSTRACT
A method of separation for an eleven component mixture comprised of 1-(2-chloroethoxy)-2-[(2-chloroethyl)thio] ethane (4) and its derivatives has been developed using LC-time-of-flight-MS. All analytical figures of merit for compounds 1-11 have been determined. Compound 4 was examined in a substrate extraction study consisting of different sand and soil matrices, and a hydrolysis study of 4 on sand revealed an extremely complex degradation pathway which appeared to be concentration dependent. Substrate extraction and hydrolysis results where compared with sulfur mustard (HD).