Bimolecular Reaction Constants Research Articles

Synergism of resistance to phosalone and comparison of kinetic properties of acetylcholinesterase from four field populations and a susceptible strain of Colorado potato beetle

The susceptibility to phosalone and biochemical characteristics of acetylcholinesterase (AChE) were compared between susceptible (SS) strain and four field populations of Colorado potato beetle (CPB) collected from commercial potato fields of Hamedan Province in west of Iran. Bioassays involving topical application of phosalone to fourth instars revealed up to 252 fold resistance in field populations compared with the SS strain. Synergism studies showed that although esterase and/or glutathione S-transferase metabolic pathways were present and active against phosalone, they were not selected for and did not have a major role in resistance. It is likely that piperonyl butoxide (PBO) reduced phosalone toxicity by inhibiting bio-activation of phosalone. The affinity ( K m) and hydrolyzing efficiency ( V max) of AChE to selected substrates, namely, acetylthiocholine iodide (ATC), propionylthiocholine iodide (PTC), and butyrylthiocholine iodide (BTC) were examined. AChE inhibition by higher substrate concentration was evident only in the SS strain. In resistant field populations, Aliabad (Aa), Bahar (B) and Dehpiaz (Dp), substrate inhibition at higher concentrations was not seen. There was no definite optimal concentration found for any of the substrates examined. When ATC, PTC, and BTC were used as substrate, the reaction rates of AChE from Yengijeh (Yg) population increased as the concentration of all three substrates were increased, but were almost constant at concentration of ATC ⩾ 3.98, PTC ⩾ 2.8, and BTC ⩾ 5 mM. The susceptible form of AChE had the most efficient ATC hydrolysis but very low BTC hydrolysis activity. In contrast, AChEs from field populations elicited relatively reduced ATC hydrolysis, but relatively increased BTC hydrolysis. The in vitro inhibition potency of some organophosphates (OPs), on AChEs of the field populations and SS strain was determined. The rank order from the most potent inhibitor to the least as determined by their bimolecular reaction constants ( K i) was ethyl paraoxon > diazoxon > methyl paraoxon for AChE from Aa, B, Dp, and Yg populations, respectively, whereas the rank order for the susceptible strain was methyl paraoxon > ethyl paraoxon > diazoxon.

Heterologous expression, purification, and biochemical characterization of a greenbug (Schizaphis graminum) acetylcholinesterase encoded by a paralogous gene (ace‐1)

Acetylcholinesterase is a critical enzyme in the regulation of cholinergic neurotransmission in insects. To produce Schizaphis graminum acetylcholinesterase-1 for structure-function analysis, we constructed a recombinant baculovirus to infect Sf9 cells, which secreted the soluble protein at a final concentration of 4.0 mg/L. The purified enzyme had an apparent M(r) of 70 and 130 kDa in the reducing and nonreducing SDS-polyacrylamide gels, respectively, indicating that it formed a dimer via an intermolecular disulfide bond. The fresh enzyme had a specific activity of 245 U/mg, which stabilized at a lower level (115 U/mg) in storage. The Michaelis constant and maximum velocity were 88.3 +/- 9.6 microM and 133.2 +/- 1.6 U/mg for acetylthiocholine iodide, 113.9 +/- 12.5 muM and 106.4 +/- 3.0 U/mg for acetyl(beta-methyl)thiocholine iodide, 68.9 +/- 7.8 microM and 76.7 +/- 1.0 U/mg for propionylthiocholine iodide, and 201.1 +/- 21.0 microM and 4.4 +/- 0.1 U/mg for S-butyrylthiocholine iodide, respectively. The IC(50) values (5 min, room temperature) of ethopropazine, BW284C51, carbaryl, eserine, malaoxon, and paraoxon were 102, 1.66, 0.94, 0.20, 0.061, 0.016 microM, respectively. The bimolecular reaction constants (k(i)) were (6.50 +/- 0.40) x 10(4) for carbaryl, (1.00 +/- 0.16) x 10(5) for eserine, (4.70 +/- 0.13) x 10(5) for malaoxon, and (9.06 +/- 0.23) x 10(5) M(-1) min(-1) for paraoxon. The enzyme was also inhibited by one of its products, choline, at concentrations higher than 20 mM, suggesting that choline bound to an anionic site and regulated the enzymatic activity.

On-chip integrated hydrolysis, fluorescent labeling, and electrophoretic separation utilized for acetylcholinesterase assay

A sensitive on-chip acetylcholinesterase (AChE) assay that serves as a basis for the development of a fully integrated on-chip AChE-inhibitor detection assay is presented. The sequential steps required for the on-chip analysis process were integrated into a microchip. Transport and mixing of the reagents occurred by a combination of electroosmosis and electrophoresis using computer-controlled electrokinetic transport. AChE-catalyzed hydrolysis of acetylthiocholine to thiocholine was determined by on-chip reaction of thiocholine with eosinmaleimide, and the resulting thioether was electrophoretically separated and detected by laser-induced fluorescence (LIF). Enzyme-substrate mixing and reaction by confluent flow of reagents was compared with electrophoretically mediated microanalysis (EMMA), based on injection of an enzyme plug, and the utilization of differences in electrophoretic mobility as a driving force for efficient mixing and reaction. Both methods yielded similar results, however the EMMA-plug technique is preferable. The EMMA-plug technique was optimized for length and pushing time of enzyme plug, length of dyes mixture plug, acetylthiocholine concentration, and detector location. Detection of O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothiolate (VX) and paraoxon, two AChE inhibitors, was demonstrated by off-chip mixing of the inhibitor and AChE, followed by the on-chip AChE assay. Limit of detection of VX for 5.5 min incubation and of paraoxon for 8 min incubation was 4 × 10 −10 and 4 × 10 −7 M, respectively. Utilization of the AChE microchip assay for inhibition kinetics was demonstrated also by evaluation of the inhibitor-enzyme bimolecular reaction constant ( k i ). The evaluated k i values for VX and paraoxon for AChE from the electric eel were 3.5 × 10 7 and 1.7 × 10 5 M −1 min −1, respectively, conforming well to reported values obtained by bulk methods.

Electron Transfer Kinetics between PQQ-dependent Soluble Glucose Dehydrogenase and Mediators

Steady-state kinetic parameters between PQQ-dependent soluble glucose dehydrogenase (PQQ-sGDH) and mediators were evaluated based on a theory of bioelectrocatalysis. Quinones showed bi-molecular reaction constants in the order of 1010 M−1 s−1 as the limiting values, while the values of Os-complexes were two or three orders smaller. The magnitude of the limiting values was interpreted in terms of the long-range electron transfer in the active center as well as the diffusion-controlled reaction model.

Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari: Ixodidae)

The San Roman strain of the southern cattle tick, Boophilus microplus, collected from Mexico was previously reported to have a high level of resistance to the organophosphate acaricide coumaphos. An oxidative detoxification mechanism was suspected to contribute to coumaphos resistance in this tick strain, as coumaphos bioassay with piperonyl butoxide (PBO) on larvae of this resistant strain resulted in enhanced coumaphos toxicity, while coumaphos assays with PBO resulted in reduced toxicity of coumaphos in a susceptible reference strain. In this study, we further analyzed the mechanism of oxidative metabolic detoxification with synergist bioassays of coroxon, the toxic metabolite of coumaphos, and the mechanism of target-site insensitivity with acetylcholinesterase (AChE) inhibition kinetics assays. Bioassays of coroxon with PBO resulted in synergism of coroxon toxicity in both the San Roman and the susceptible reference strains. The synergism ratio of PBO on coroxon in the resistant strain was 4.5 times that of the susceptible strain. The results suggested that the cytP450-based metabolic detoxification existed in both resistant and susceptible strains, but its activity was significantly enhanced in the resistant strain. Comparisons of AChE activity and inhibition kinetics by coroxon in both susceptible and resistant strains revealed that the resistant San Roman strain had an insensitive AChE, with a reduced phosphorylation rate, resulting in a reduced bimolecular reaction constant. These data indicate a mechanism of coumaphos resistance in the San Roman strain that involves both insensitive AChE and enhanced cytP450-based metabolic detoxification.

Comparative distribution and kinetic properties of acetylcholinesterase from Apis mellifera and Apis cerana

SUMMARYWe determined the distribution of acetylcholinesterase (AChE) activities in extracts from the head, thorax and abdomen of Apis cerana cerana and Apis mellifera ligustica. There was no statistical difference in the AChE activity from the head or abdomen between the two honey bee species. However, AChE activity in the thorax was significantly greater (P < 0.01) in A. cerana than in A. mellifera. The substrate specificity and the sensitivity to inhibitors of AChEs from the head of two species were studied using acetylthiocholine iodide (ATCh), acetyl-(β-methyl) thiocholine (MeTCh), s-propionylthiocholine (PrTCh) and s-butyrylthiocholine iodide (BuTCh) as substrates and eserine, propoxur, thiodicarb and methomyl as inhibitors, respectively. There was no statistical difference in AChE affinities toward the four substrates between A. mellifera and A. cerana based on comparison of the Michaelis constant (Km) values. However, the hydrolyzing efficiency of AChE from A. mellifera for four substrates was significantly higher than that from A. cerana. Among the tested inhibitors, eserine was the most potent inhibitor to AChEs of both bees; the bimolecular reaction constant (k1 was 400.3 for A. mellifera and 340.3 for A. cerana. Responses of AChE to carbamate insecticides differed for the bees. For A. mellifera, kis were: propoxur = 5.7, thiodicarb = 5.2 and methomyl = 4.9. For A. cerana, k1s were: methomyl = 10.4, propoxur = 4.6 and thiodicarb = 4.2. AChE from A. mellifera thus was more sensitive to inhibitors than AChE from A. cerana.

Comparative inhibition kinetics for acetylcholinesterases extracted from organophosphate resistant and susceptible strains of Boophilus microplus (Acari: Ixodidae).

In this study, acetylcholinesterases (AChEs) were extracted from two Mexican Boophilus microplus strains that demonstrated resistance to the organophosphate (OP) acaricide, coumaphos, in bioassay. The rate of inhibition of the extracted AChEs by the diethyl-OP paraoxon was determined for two resistant strains and two susceptible strains of B. microplus. The time to inhibition of 50% AChE activity was approximately two-fold greater for the resistant strains. Kinetic analysis of the interaction of the resistant AChEs with paraoxon revealed reduced bimolecular reaction constants (ki). Apparent conformational changes in the AChE of the resistant strains were reflected in reduced Km and Vmax values. The bimolecular reaction constants (ki) of the resistant strains were most affected by a slower rate of enzyme phosphorylation (k2).

Determination of the nucleophilic reactivity constants for a series of N-( n-propyl)- N′-( para- R-benzoyl)thioureas towards trans-[Pt(pyridine) 2Cl 2

The relative nucleophilic reaction constants n pt, for a series of N-( n-propyl)- N′-( para- R-benzoyl)thioureas (R=H, CH 3O, (CH 3) 3C, Br, Cl, C 7H 15O, I, NO 2) have been determined for the substrate trans-[PtCl 2(pyridine) 2] in methanol–water (75:25 v/v at 30 °C and μ=0.03), and are compared with that of unsubstituted thiourea. The bimolecular substitution reaction constants k 2 were found to vary from 12.1±1.3 to 6.0±0.4 (for thiourea), corresponding to relative nucleophilicity constants of 4.08–3.78. A small but detectable influence of the substituent has been found on these constants, the n pt values decreasing in the order H∼CH 3O>Cl>C 7H 15O∼t-but∼I>Br>NO 2>thiourea. Essentially however, the nucleophilicity of the series of N-( n-propyl)- N′-( para- R-benzoyl)-thioureas resembles that of thiourea.

Oxidation of melatonin by oxoferryl hemoglobin: A mechanistic study

Reaction of melatonin with the hypervalent iron centre of oxoferryl hemoglobin, produced in aqueous solution from methemoglobin and H2O2, has been investigated at 37°C and pH 7.4, by absorption spectroscopy. The reaction results in reduction of the oxoferryl moiety with formation of a heme-ferric containing hemoprotein. Stopped-flow spectrophotometric measurements provide evidence that the reduction of oxoferryl-Hb by melatonin is first-order in oxoferryl-Hb and first-order in melatonin. The bimolecular reaction constant at pH 7.4 and 37°C is 112 ± 1.0 M-1 s-1.Two major oxidation products from melatonin have been found by gas chromatography-mass spectroscopy: the cyclic compound 1,2,3,3a,8,8a-hexahydro-1-acetyl-5-methoxy-3a-hydroxypyrrolo[2,3-b]indole (cyclic 3-hydroxy-melatonin), and N-acetyl-N′-formyl 5-methoxykynuramine (AFMK). The percentage yield of the two major products appears dependent on the ratio [oxoferryl-Hb]: [melatonin]—the higher the ratio the higher the yield of AFMK. The observed stoichiometry oxoferryl-Hbreduced:melatoninconsumed is 2, when the ratio [oxoferryl-Hb]:[melatonin] is 1:1, but appears >2 at higher molar ratios. The reduction of the hypervalent iron of the oxoferryl moiety may be consistent with an oxidation of melatonin by two one-electron steps.

Irreversible inhibition of nicotinic acetylcholine receptors by the bipinnatins. Toxin activation and kinetics of receptor inhibition.

Bipinnatin-A, -B, and -C belong to a family of naturally occurring marine neurotoxins known as the lophotoxins. The lophotoxins are unique in that they irreversibly inhibit nicotinic acetylcholine receptors by forming a covalent bond with a tyrosine residue at position 190 in the alpha-subunit of the receptor. In this study, we show that the inhibitory activity of the bipinnatins against the nicotinic receptor increased with preincubation of the toxins in aqueous buffer prior to incubation with the receptor. The parent species of the bipinnatins displayed little, if any, affinity for the nicotinic receptor. Preincubation of the toxins appeared to produce a single, relatively stable, active toxin species that irreversibly inhibited the two acetylcholine-binding sites on the nicotinic receptor with two distinguishable apparent pseudo first-order rates. The difference in the rates of irreversible inhibition of the two binding sites on the receptor was exploited to selectively inhibit one site for the pharmacological investigation of the other. The bipinnatins preferentially inhibited the binding site near the alpha/delta-subunit interface that displays low affinity for metocurine and high affinity for acetylcholine. The bimolecular reaction constants for the interaction of the bipinnatins with the nicotinic receptor decreased in the order bipinnatin-B > bipinnatin-A > bipinnatin-C for both acetylcholine-binding sites. The ratio of the bimolecular reaction constants for the two binding sites on the receptor was not the same for the three bipinnatins. This indicates that the reaction of the bipinnatins with the nicotinic receptor is sensitive to differences in the structure of the two acetylcholine-binding sites. The bipinnatins may be useful in the design of novel drugs for the nicotinic receptor that exclusively inhibit one of the two binding sites and for the investigation of structural differences between the two acetylcholine-binding sites of the receptor.

Acetylcholinesterase from the horn fly (Diptera: Muscidae) II: Biochemical and molecular properties

AbstractPurified acetylcholinesterase (AChE) of the horn fly was characterized to elucidate the enzymological, inhibitory, and molecular properties of the enzyme. Maximum activity of the AChE against the substrate acetylthiocholine (ATCh) occurred when reactions were conducted at 37°C and pH 7.5. Km and Vmax values were (9.2 ± 0.35) × 10−6 M and 239.8 ± 10.8 units/mg, respectively, for ATCh and (1.5 ± 0.07) × 10−5 M and 138.5 ± 5.5 units/mg, respectively, for butyrylthiocholine (BTCh). The activity of AChE decreased when concentrations of ATCh or BTCh were higher than 1 mM. Studies of the interaction of AChE with different inhibitors revealed pl50 values of 8.88 for eserine, 6.90 for BW284C51, and 4.97 for ethopropazine. Bimolecular reaction constants (kis) for the organophosphorus (OP) anticholinesterases were (2.74 ± 0.14) × 106 M−1 min−1 for coroxon, (7.20 ± 0.28) × 105 M−1 min−1 for paraoxon, and (2.33 ± 0.12) × 105 M−1 min−1 for stirofos. Two major forms of native AChE molecules were found on non‐denaturing polyacrylamide gel electrophoresis (PAGE) with Triton X‐100, corresponding to bands AChE‐2 and AChE‐4 found on PAGE without Triton X‐100. AChE‐2 had an estimated molecular weight of 603,000 and was amphiphilic. AChE‐4 had a molecular weight of 147,000 and was hydrophilic. Results of PAGE analyses indicated that the purified enzyme had two bands, one of about 123 kDa and the other greater than 320 kDa, prior to disulfide reduction and only one band at about 54 kDa after reduction on SDS‐PAGE. © 1994 Wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.

A potentiometric biosensor for pesticides based on the thiocholine hexacyanoferrate (III) reaction

A solid-state acetylcholinesterase (AChE) sensor was developed on the basis of AChE acetylthiocholine-hexacyanoferrate (III) reaction. Various properties of the sensor were investigated and possibilities for its practical use in the detection and assay of pesticides are suggested. The response of the sensor was induced by two phenomena: the Donnan effect and the redox potential. Unlike pH-sensitive sensors, this type of sensor is operated at high ionic strength and a high buffer capacity. The ability of the AChE sensor to assay pesticides was demonstrated by the characterization and quantitative determination of dichlorvos based on its inhibition of AChE. The kinetics of inhibition by dichlorvos of the reaction with AChE were studied by potentiometric and photometric methods. The bimolecular reaction constant was 7·07 10 5 M −1 min −1 and the affinity constant was 4·76 10 −6 M. The detection limits for dichlorvos were 1 μM for a flow-injection mode and 0·2 μM for stationary measurement. The AChE acetylthiocholine-hexacyanoferrate(III) system opens up possibilities for the detection and assay of toxic chemicals by the use not only potentiometric but also of other types of sensors.

Enzymological and inhibitory properties of acetylcholinesterase purified from Lygus hesperus knight (Hemiptera:Miridae)

We studied the enzymological and inhibitory properties of a 600-fold purified acetylcholinesterase (AChE, EC 3.1.1.7) from Lygus hesperus Knight. The optimal pH and temperature were 7.5–8.0 and 35–40°C, respectively, for hydrolyzing selected substrates. K m and V max were 6.9 × 10 −5 M and 42.9 units/mg for acetylthiocholine (ATC), 6.5 × 10 −5 M and 32.8 units/mg for acetyl-(β-methyl) thiocholine (AβMTC), 7.2 × 10 −5 M and 21.9 units/mg for propionylthiocholine (PTC), and 3.3 × 10 −3 M and 4.2 units/mg for S-butyrylthiocholine (BTC), at 37°C and pH 7.7. The efficiency for hydrolyzing different substrates was ATC > A βMTC > PTC > BTC. Enzyme activity was inhibited by 10 −7 M eserine or BW284C51, but not by ethopropazine. The enzyme's inhibitor and substrate specificities, and substrate (ATC) inhibition confirmed that it was an AChE. Bimolecular reaction constants were 9.44 × 10 5 M −1 min −1 for paraoxon, 2.21 × 10 5 M −1 min −1 for dichlorvos, 8.28 × 10 6 M −1 min −1 for naled, 2.15 × 10 4 M −1 min −1 for oxydemeton-methyl, 4.18 × 10 4 M −1 min − for demeton- S-methyl, for 2.98 × 10 5 M −1 min −1 for mevinphos, at 25°C. The inhibitory potency for all six organophosphorus compounds was mainly determined by the affinity between the organophosphates and AChE. Eserine and carbofuran were competitive inhibitors for the active sites of AChE. The competitive inhibition constant was 1.20 × 10 −8 M for eserine and 1.24 × 10 −8 M for carbofuran.

Chiral reactions of acetylcholinesterase probed with enantiomeric methylphosphonothioates: Noncovalent determinants of enzyme chirality

Abstract Enantiomeric cycloheptyl- and isopropyl methylphosphonothioates containing uncharged and cationic leaving groups, and 3,3-dimethylbutyl methylphosphonyl thiocholines were synthesized, and their inhibition of acetylcholinesterase from Torpedo examined. Bimolecular inhibition constants spanned 10(1)-10(9) M-1.min-1, equilibrium dissociation constants 10(-3)-10(-7) M, and phosphonylation constants 1-300 min-1. A general but not absolute preference for the SP-enantiomer, in the range 170-4600 for cycloheptyl-, 0.6-150 for isopropyl-, and 30 for 3,3-dimethylbutyl methylphosphonothioates, varied with nature of the alkyl ester (-OR) and thioic leaving groups (-SR') surrounding phosphorus. While the overall bimolecular reaction constant showed no marked dependence on ionic strength of the medium, the microscopic kp and KD for the RP- but not SP-cycloheptyl methylphosphonyl thiocholine underwent marked reduction with decreases in ionic strength. This result unmasks the interplay between occupation of the active center and productivity of that occupation. These studies reveal that chiral reactions with acetylcholinesterase are dependent more on the nature of the groups surrounding the tetrahedral phosphorus than on the absolute configuration about the phosphorus atom and indicate that the active center comprises partially overlapping subsites that can accommodate the -OR and -SR' groups. The presence of neighboring subsites characterized by different steric, electrostatic, and hydrophobic properties permits a multiplicity of binding orientations, independent of chiral configuration, and which account for the large variation in chiral preference seen among organophosphonates containing different substituents.

Mechanism of agonist and antagonist binding to alpha 2 adrenergic receptors: evidence for a precoupled receptor-guanine nucleotide protein complex.

The alpha 2 adrenergic receptor (AR) inhibits adenylate cyclase via an interaction with Ni, a guanine nucleotide binding protein. The early steps involved in the activation of the alpha 2 AR by agonists and the subsequent interaction with Ni are poorly understood. In order to better characterize these processes, we have studied the kinetics of ligand binding to the alpha 2 AR in human platelet membranes on the second time scale. Binding of the alpha 2 antagonist [3H]yohimbine was formally consistent with a simple bimolecular reaction mechanism with an association rate constant of 2.5 X 10(5) M-1 s-1 and a dissociation rate constant of 1.11 X 10(-3) s-1. The low association rate constant suggests that this is not a diffusion-limited reaction. Equilibrium binding of the alpha 2 adrenergic full agonist [3H]UK 14,304 was characterized by two binding affinities: Kd1 = 0.3-0.6 nM and Kd2 = 10 nM. The high-affinity binding corresponds to approximately 65% and the low-affinity binding to 35% of the total binding. The kinetics of binding of [3H]UK 14,304 were complex and not consistent with a mass action interaction at one or more independent binding sites. The dependence of the kinetics on [3H]UK 14,304 concentration revealed a fast phase with an apparent bimolecular reaction constant kappa + of 5 X 10(6) M-1 s-1. The rate constants and amplitudes of the slow phase of agonist binding were relatively independent of ligand concentration. These results were analyzed quantitatively according to several variants of the "ternary complex" binding mechanism. In the model which best accounted for the data, (1) approximately one-third of the alpha 2 adrenergic receptor binds agonist with low affinity and is unable to couple with a guanine nucleotide binding protein (N protein), (2) approximately one-third is coupled to the N protein prior to agonist binding, and (3) the remainder interacts by a diffusional coupling of the alpha 2 AR with the N protein or a slow, ligand-independent conformational change of the alpha 2 AR-N protein complex. The rates of interaction of liganded and unliganded receptor with N protein are estimated.

Eel acetylcholinesterase inhibition studies with heteroarylphosphinates

The inhibition of eel acetylcholinesterase by the 4-nitrophenyl esters of 2-furyl(methyl)-, methyl(2-thienyl)-, di-2-furyl-, and di-2-thienylphosphinic acid (I, II, III, and IV, respectively) was investigated at pH 6.90 in 0.067 M phosphate buffer (25.0°C) using stopped-flow instrumentation and automated data processing. Our evaluation of the dissociation constant, K d , the unimolecular bonding rate constant, k 2, and the bimolecular reaction constant, k i , are the first reported values for these constants for alkyl/heteroaryl and diheteroaryl esters of phosphinic acids. The largest k i value (19,330 M −1 sec −1) was observed for the reaction of I with the enzyme. The order for the remaining three is II > IV > III. There is no direct relationship between the hydrolysis rates of the esters and their anticholinesterase activities on eel acetylcholinesterase. Likewise, there is no direct relationship between their anticholinesterase activities and the LD 50 values in rats.

Inhibition of two acetylcholinesterases by the 4-nitrophenyl esters of methyl-, ethyl-, and isopropyl(phenyl)phosphinic acid

The inhibition of eel acetylcholinesterase and bovine erythrocyte acetylcholinesterase by the 4-nitrophenyl esters of methyl-, ethyl-, and isopropyl(phenyl)phosphinic acid (MPP, EPP, and IPP, respectively) was investigated at pH 6.90 in 0.067 M phosphate buffer (25.0°C) using stopped-flow instrumentation and automated data processing. Our evaluation of the dissociation constant, K d , the unimolecular bonding rate constant, k 2, and the bimolecular reaction constant, k i , are the first reported values for these constants for a homologous series of this class of organophosphorus compounds. The largest k 1 value (29,428 M −1 sec −1) was observed for the reaction of eel acetylcholinesterase with 4-nitrophenyl methyl(phenyl)phosphinate. The smallest k i value (9.6 M −1 sec −1) was observed for the reaction of bovine erythrocyte acetylcholinesterase with 4-nitrophenyl isopropyl(phenyl)phosphinate.

Dissociation and phosphorylation constants for the inhibition of acetylcholinesterase by 2-fluoro, 2-oxo-1,3,2-dioxaphosphorinanes

Phosphorylation, dissociation and bimolecular reaction constants were determined for a number of fluoro 1,3,2-dioxaphosphorinane 2-oxide derivatives in the presence of substrate. The data are discussed in terms of the steric effects of dioxaphosphorinane ring substituents on the kinetic and dissociation constants for the inhibition reaction.

Studies on the inhibition of honey bee cholinesterase by carbamates

The kinetic constants for the inhibition (I50, Ka, K2c, -Ki, K3, and Ka' values) of honey bee cholinesterase by furadan and its two analogues were determined and evaluated. Furadan proved to be a potent inhibitor of honey bee enzyme. All the three carbamates showed a high affinity for honey-bee ChE. The affinity constants ranged from 0.22 to 2 .6 8 x 1 0 -- M. There was little variation among the carbamylation constants, indicating a minor role for the carbamylation step in the inhibition process. The significant variation in the bimolecular reaction constants of the three carbamates could be attributed to the variations in the affinity constants rather than to the carbamylation constants. In most cases, the average apparent affinity constants were comparable with the Ka values and increased progressively with the concentration of inhibitor.

Relation of acetylcholinesterase sensitivity to cross-resistance of a resistant house fly strain to organophosphates and carbamates

Acetylcholinesterase (AChE, E.C. 3.1.1.7) from an organophosphate-resistant strain of house fly, Musca domestica (L.) exhibited a decrease in sensitivity towards four organophosphates and two carbamates in comparison with enzyme from the parent susceptible strain. Sensitivity was less, as measured by the bimolecular reaction constant ( k i), by a factor of 117 for dichlorvos, 94 for paraoxon, 11 for diazoxon, 7 for Tetram, 62 for propoxur, and 50 for dimetilan. These differences in bimolecular reaction constants were attributed entirely to differences in their affinity for the enzyme, as measured by the dissociation constant, K d . It is suggested that the cross resistance to these inhibitors is due at least in part to insensitive acetylcholinesterase.