ABSTRACT
Organophosphorus hydrolase (OPH) is capable of hydrolyzing a wide variety of organophosphorus pesticides and chemical warfare agents. However, the hydrolytic activity of OPH against the warfare agent VX is less than 0.1% relative to its activity against parathion and paraoxon. Based on the crystal structure of OPH and the similarities it shares with acetylcholinesterase, eight OPH mutants were constructed with the goal of increasing OPH activity toward VX. The activities of crude extracts from these mutants were measured using VX, demeton-S methyl, diisopropylfluoro-phosphate, ethyl parathion, paraoxon, and EPN as substrates. One mutant (L136Y) displayed a 33% increase in the relative VX hydrolysis rate compared to wild type enzyme. The other seven mutations resulted in 55-76% decreases in the relative rates of VX hydrolysis. There was no apparent relationship between the hydrolysis rates of VX and the rates of the other organophosphorus compounds tested.
Subject(s)
Chemical Warfare Agents/pharmacokinetics , Esterases/chemistry , Esterases/metabolism , Insecticides/pharmacokinetics , Organothiophosphorus Compounds/pharmacokinetics , Acetylcholinesterase/chemistry , Amino Acid Substitution , Aryldialkylphosphatase , Base Sequence , Biodegradation, Environmental , DNA Primers , Hydrolysis , Isoflurophate/pharmacokinetics , Mutagenesis, Site-Directed , Organothiophosphates/pharmacokinetics , Paraoxon/pharmacokinetics , Parathion/pharmacokinetics , Phenylphosphonothioic Acid, 2-Ethyl 2-(4-Nitrophenyl) Ester/pharmacokinetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
Herein a molecular mechanic study of the interaction of a lethal chemical warfare agent, O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (also called VX), with Torpedo californica acetylcholinesterase (TcAChE) is discussed. This compound inhibits the enzyme by phosphonylating the active site serine. The chirality of the phosphorus atom induces an enantiomeric inhibitory effect resulting in an enhanced anticholinesterasic activity of the SP isomer (VXS) versus its RP counterpart (VXR). As formation of the enzyme-inhibitor Michaelis complex is known to be a crucial step in the inhibitory pathway, this complex was addressed by stochastic boundary molecular dynamics and quantum mechanical calculations. For this purpose two models of interaction were analyzed: in the first, the leaving group of VX was oriented toward the anionic subsite of TcAChE, in a similar way as it has been suggested for the natural substrate acetylcholine; in the second, it was oriented toward the gorge entrance, placing the active site serine in a suitable position for a backside attack on the phosphorus atom. This last model was consistent with experimental data related to the high inhibitory effect of this compound and the difference in activity observed for the two enantiomers.
Subject(s)
Acetylcholinesterase/chemistry , Chemical Warfare Agents/chemistry , Cholinesterase Inhibitors/chemistry , Organothiophosphorus Compounds/chemistry , Acetylcholinesterase/metabolism , Animals , Binding Sites , Chemical Warfare Agents/metabolism , Cholinesterase Inhibitors/metabolism , Molecular Structure , Organothiophosphorus Compounds/metabolism , Serine/chemistry , TorpedoABSTRACT
Synthesis of new bis(1-methylpyridinium) compounds containing a 1,4-diacetylbenzene linkage between the pyridinium moieties from commercially available 2-, 3-, and 4-picoline precursors was accomplished via metallation, reaction of the picolyllithium with 1,4-dicyanobenzene, and subsequent quaternization of the resulting bispyridyl compounds. Acetylcholinesterase inhibitory activity was determined colorimetrically with purified electric eel enzyme. Examination of structure-activity relationships indicated that the 3-substituted pyridinium compound is the most potent isomer, followed by the 2-substituted isomer, and that the 4-substituted analogue is the least active.
Subject(s)
Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/pharmacology , Pyridinium Compounds/chemical synthesis , Pyridinium Compounds/pharmacology , Animals , Electrophorus , Molecular Conformation , Structure-Activity RelationshipABSTRACT
The yeast ERG2 gene codes for the C-8 sterol isomerase, an enzyme required for the isomerization of the delta 8 double bond to the delta 7 position in ergosterol biosynthesis. The ERG2 gene was cloned by complementation of a C-8 sterol isomerase mutant strain (erg2). The complementing region of DNA required to restore ergosterol synthesis to erg2 was limited to a 1.0 kb StuI-BglII fragment. In order to determine whether the ERG2 gene was essential for yeast viability, a LEU2 gene was inserted into the NdeI site (made blunt) of this 1.0 kb fragment. Transformation of a wild type diploid strain with the ERG2 substituted DNA resulted in the generation of viable haploids containing the erg2 null allele (erg2-4::Leu2). These results suggest that the C-8 sterol isomerase activity is not essential for yeast cell viability. This disruption represents the second ergosterol biosynthetic gene in the distal portion of the pathway to be disrupted without adversely affecting cell viability.
Subject(s)
Cloning, Molecular , Saccharomyces cerevisiae/genetics , Steroid Isomerases/genetics , Ergosterol/biosynthesis , Genes, Fungal , Haploidy , Mutagenesis , Restriction Mapping , Saccharomyces cerevisiae/enzymology , Transfection , Transformation, GeneticABSTRACT
Organophosphorus compounds have been shown to exhibit toxic behavior as insecticides, pesticides, and mammalicides. Soman and 21 related compounds were studied for possible structure-activity relationships. Computer-aided methods were used to generate a linear expression relating the activity (ln [1/LD50], rabbit I.V.) of the compounds to three structure-based descriptors (R = 0.96). Principal components regression and jackknife analysis were performed to assess the stability of the model.
