TOXICOLOGY AND APPLIED PHARMACOLOGY 91, 140- 144 ( 1987)

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Kinetic Constants for the Inhibition of Eel and Rabbit Brain Acetylcholinesterase by Some Organophosphates and

Carbamates of Military Significance

Kinetic Constants for the Inhibition of Eel and Rabbit Brain Acetylcholinesterase by Some Organophosphates and Carbamates of Military Significance. GRAY P. J., AND DAWSON, R. M.

(1987). Toxicol. Appl. Pharmacol. 91, 140-144. The kinetics of the inhibition of eel and rabbit brain acetylcholinesterase by a number of organophosphates and carbamates has been studied. Five organophosphates (tabun, satin, soman, GF, and VX) and two carbamates (neostigmine and physostigmine) were used. Small but significant differences have been found between the two enzyme preparations in both the dissociation constant and the rate constants for the irrevers- ible step. The values of k2 ranged from 0.56 to 1.08 see-’ for the eel enzyme and 0.19 to 0.73 sec.-’ for the rabbit enzyme. Kd varied from 0.3 to 24.5 pM for the eel and 0.3 to 9.3 fiM for the rabbit enzyme. The similarity between the enzymes is remarkable considering the differences in the Speck3 of origin. 0 1987 Academic Press. Inc.

The inhibition of acetylcholinesterase (acetyl- choline acetylhydrolase; EC 3.1.1.7; AChE) by organophosphorus and carbamate in- hibitors occurs in two stages as indicated by Eq. (1).

kl k2 EH+IXLF EH.IX -

1

EI+HX;oEH+IOH, (1) 2

where EH is the enzyme and IX the inhibitor. Inhibition proceeds by formation of a revers- ible enzyme-inhibitor complex (EH . IX) fol- lowed by formation of the enzyme-phospho- rus or enzyme-carbon bond with displace- ment of the leaving group. The reaction may be described by the dissociation constant Kd (k-,/k,), the unimolecular rate constant k2, and the reactivation rate constant k,; k3 is ob- served to be much smaller than k2. The over- all inhibitory power of the inhibitor is usually expressed as ki = k2 fKd.

Electric eel acetylcholinesterase has been one of the most commonly used enzymes for the study of the mechanism of action of these compounds. However, this enzyme is not a suitable model for mammalian erythrocyte

and brain AChE (Dawson and Poretski, 1985; De Jong and Wohing, 1984). Several studies have compared the effects of organo- phosphorus insecticides on AChE from a range of mammalian and nonmammalian species (Wang and Murphy, 1982; Fulton and Chambers, 1985). However, these inhibi- tors are much less potent than the nerve agents. Andersen et al. (1977) found large differences in the values of ki determined for the inhibition of AChE from several species by sarin, soman, and tabun. However, the values of k2 and Kd were not reported. In view of these considerations and the question of a correlation between Kd and k2 (For&erg and Puu, 1984), it is of interest to determine these parameters for the nerve agents in a mamma- lian species and compare the values with those obtained for the eel enzyme under the same conditions.

This paper reports values of k2 and Kd for the potential chemical warfare agents tabun, sarin, soman, GF, and VX and also for the carbamates neostigmine and physostigmine. AChE from both electric eel and rabbit brain were used.

METHODS Muferiuls. Small amounts of the organophosphorus

compounds were synthesized in the Organic Chemistry

0041-008X/87 $3.00 Copyright 0 1987 by Academic Press, Inc. All rigbt.9 of re~oduction in any fOt’m mr~ed.

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Division, Materials Research Laboratories, and were greater than 95% pure. They are tabun (ethyl N,Ndi- methylphosphoramidocyanate); satin (isopropylmethyl- phosphonofluoridate); soman (pinacolylmethylphos- phonofluoridate); GF (cyclohexylmethylphosphono- fluoridate), and VX (O-ethyl 42-diisopropylaminoethyl methyl phosphonothiolate).

Physostigmine and neostigmine were obtained from Calbiochem-Behring and K & K Laboratories, respec- tively. Eel AChE (type VI-S, 340-350 units/mg), acetyl- thiocholine iodide (ASChI), and 5,5’-dithiobis (2-nitro- benzoic acid) (DTNB) were obtained from Sigma Chem- ical Co. Rabbit brain AChE, solubilized with Triton X- 100, was prepared and partially purified by affinity chro- matography as described by Dawson and Poretski (1985).

Stopped-flow method. In order to measure k2 and Kd

accurately, it is necessary to use concentrations of inhibi- tor approaching or exceeding Kd. For organophosphate and carbamate inhibitors of AChE, the magnitude of k2 means that under these conditions complete inhibition may occur in a matter of seconds (Hart and O’Brien, 1973). Such reactions are best followed by using a stopped-flow apparatus and in the presence of substrate (Hart and O’Brien, 1973; Horton et al., 1977; Forsberg and Puu, 1984).

All the experiments were carried out at 25°C with a modified Aminco-Morrow stopped-flow apparatus. The reaction of DTNB with the substrate hydrolysis product thiocholine produced a yellow thionitrobenzoate ion. Production of this anion was monitored at 4 12 nm using a blue-enhanced photodiode (Silicon Detector Corp.). The diode output was amplified, digitized, stored on a floppy disk for later analysis, and simultaneously dis- played on a chart recorder. The detailed construction of the apparatus may be found in Gray (1985).

All experiments were carried out in 0.067 M phosphate buffer, pH 7.0, prepared in deionized water. A stock solu- tion of 0.2 mg/ml eel AChE (lyophilized powder) in buffer containing 1 .O mg/ml gelatin was frozen in small aliquots. The working enzyme solution was prepared by diluting the stock enzyme 250-fold in buffer containing 0.04 mg/ml gelatin. The substrate solution contained ASChI (2 mM), DTNB (0. I mM), and inhibitor. The mix- ing process resulted in a 2-fold dilution of both solutions. Stock solutions of nerve agents were prepared at a con- centration of 5 mg/ml in ethanol and added to the sub- strate immediately before use. The final ethanol concen- tration had no observable effect on the AChE. Stockcar- bamate solutions were 0.01 M in buffer and were also added to the substrate immediately before use. The rab- bit brain AChE (1.2 mg protein/ml) was stored frozen and diluted 12-fold in 0.067 M phosphate buffer. The buffer did not contain gelatin and the final concentra- tions of Triton X-100, Tris, and NaCl were 0.002% and 0.02 and 4 mM, respectively.

The uninhibited rates under these conditions were 4.8 x IO-’ mol/sec for the rabbit brain enzyme and 1.4 x 1 Oe6 mol/sec for the eel enzyme. The K,,, values were 7.3 X 10-j and 5.9 X 10-j M for the eel and rabbit brain enzymes, respectively.

Data analysis. The constants Kd and k2 were derived from replicate kinetic experiments, coupled with a con- trol experiment to determine the rate of substrate hydro- lysis, V,, in the absence of inhibitor. The equations (Hor- ton et al., 1977) are

Km [=I Kd=(K,+[S])(V,/VO- 1) (2)

where VO and V, are the rates of substrate hydrolysis in the presence of inhibitor (IX) at times zero and t, respec- tively. [S] is the substrate concentration and K,,, the Mi- chaelis constant.

Further details of the experimental procedure and analysis have been published by Gray (1985).

RESULTS AND DISCUSSION

The kinetic constants for the inhibitors are given in Table 1. The results are presented as the means and standard deviations of experi- ments carried out at the highest inhibitor concentration commensurate with an ade- quate absorbance change. The constants show that the inhibitory power (ki) of a nerve agent or carbamate is determined by its affinity for the enzyme (Kd), while k2 varies little from one agent to another. Thus, for eel AChE, Kd varies by a factor of 82 between ta- bun (24.5 PM) and GF (0.3 PM) while k2 var- ies by a factor of 1.4 between the same agents. Similarly, the rabbit brain enzyme Kd varies by a factor of 27 between tabun (8.2 PM) and GF (0.3 PM) while k2 varies by a factor of 1.3.

A comparison of the results for the rabbit and eel enzymes (Table 1) shows that the rank order of the ki values for the nerve agents is the same for both enzymes. Application of the Student t test at the 5% level of signifi- cance yields the following information for the nerve agents:

1. The values of k2 for the rabbit enzyme are all significantly lower than the values for

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TABLE 1

KINETICCONSTANTSFORTHEINHIBITIONOFEELANDRABBITBRAINACETYLCHOLINESTERASEBYSOME ORGANOPHOSPHORUSCOMPOUNDSANDCARBAMATES

Inhibitor Concentration (M) k2 (set-‘) Kd (PM) k, (M--l set -‘)

Eel AChE

Tabun 1.6 x lO-4 0.78(0.08) 24.5(0.2) 3.2 x IO4 Sarin 1.1 x 10-r 1.05 (0.10) 2.2 (0.2) 4.8 x IO5 Soman 2.6 x lO-6 0.92(0.10) 0.4(0.03) 2.3 x 10’ GF 1.2 x 10-6 1.08 (0.22) 0.3(0.1) 3.6 x 10" vx 9.2 x lO-6 0.92(0.18) 1.7(0.4) 5.4 x lo5

Neostigmine 1.0 x 10-4 0.56(0.04) 8.8(0.4) 6.6 x IO4 Physostigmine 6.3 x lo-’ 0.59(0.06) 7.1 (1.0) 8.3 x 104

Rabbit Brain AChE

Tabun 3.3 x 10-5 0.56(0.05) 8.2(1.2) 6.8 x lo4 Satin 7.4 x lo-6 0.42(0.12) 1.7 (0.5) 2.5 x IO5 Soman 1.9 x 10-6 0.45(0.05) 0.3(0.04) 1.5 x lo* GF 5.8 x lo-’ 0.73(0.18) 0.3(0.1) 2.4 x 10” vx 2.7 x 10m6 0.5 1 (0.08) 0.9(0.2) 5.7 x 10’ Neostigmine 2.5 x lO-5 0.62(0.10) 9.3(1.1) 6.7 x IO4 Physostigmine 2.5 x lO-5 0.19 (0.03) 2.7(0.6) 7.0 x IO4

Note. The experiments were carried out at 25°C in 0.067 M phosphate buffer, pH 7.0. The standard deviations are given in parentheses. The average number of replicates was 13.

the eel AChE. The differences, although sta- tistically significant, are small.

2. With the exception of GF (no signifi- cant difference) the values of Kd for the rabbit enzyme are also statistically significantly lower than the values for the eel AChE.

3. The ki values for satin, soman, and GF are significantly lower, for VX not signifi- cantly different, and for tabun significantly higher for rabbit brain than for the eel en- zyme.

The kinetic constants for neostigmine were not significantly different between the two en- zymes. However, for physostigmine, the val- ues obtained for the rabbit enzyme were sig- nificantly lower than for the eel enzyme.

The stopped-flow technique described above makes advantageous use of acetylthio- choline as substrate. Previous workers have used pnitrophenylacetate (Hart and O’Brien, 1973, 1974; For&erg and Puu, 1984). When

using p-nitrophenylacetate, it is necessary to correct for a residual hydrolysis rate which occurs when the enzyme is completely inhib- ited (Hart and O’Brien, 1974; Horton et al., 1977). No such corrections were required for acetylthiocholine (Gray, 1985).

The kinetic parameters for eel AChE are in good agreement with those of For&erg and Puu (1984) for the three nerve agents that they used (sarin, soman, and VX). The results for rabbit brain AChE are qualitatively the same as for the eel enzyme in that the Kd var- ies by a large factor while the k2 remains al- most constant. However, there are significant differences between the constants for the rab- bit and eel enzymes, the values of Kd and k2 being lower for the rabbit than the eel en- zyme. The differences observed, although statistically significant, are small compared with the species differences observed for the organophosphorus insecticides (Wang and Murphy, 1982; Fulton and Chambers, 1985)

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and smaller than the differences observed by Andersen et al. (1977) for the nerve agents and species they studied.

The results extend those of Forsberg and Puu (1984) who did not report on GA and GF. The data on GA are of particular interest since it is the only organophosphate for which there is substantial evidence of actual use as a chemical weapon (United Nations Security Council S/ 16433,1984). The similarity of the results for GD and GF is not surprising con- sidering the similarity of their structures. GB also has the same basic structure, but a smaller alkyl group (isopropyl). The much lower inhibitory power that GB exhibits em- phasizes the importance of the size of this group in inhibitory power as well as in aging.

The question of a correlation between Kd and k2 has been raised by others (For&erg and Putt, 1984). The approach used thus far has been to compare a range of compounds using the same enzyme (Forsberg and Putt, 1984). Our data, which compare the same compounds with two different active sites, provide some support for such a correlation. This is because in every case (for both nerve agents and carbamates) in which the Kd was lower or higher for one enzyme than the other, the kZ was correspondingly lower or higher.

The observation that the ki of each agent depends mainly on its affinity for the enzyme (Kd) whereas k2 varies little from one inhibi- tor to another suggests that any means of re- ducing k2 for one inhibitor may well have a similar effect on all other inhibitors of the same group. The use of allosteric effecters may provide such a mechanism for reducing kZ. Several allosteric effecters are known which influence the catalytic activity of AChE by binding to a site(s) peripheral to the active site (Tomlinson et al., 1980). The properties of AChE which are affected by binding to an allosteric site are the rates of carbamylation, decarbamylation, aging of or- ganophosphate-inhibited AChE, and sub- strate hydrolysis (Dawson et al., 1981). Al-

though relatively high concentrations of allo- steric effector are required in vitro for observable effects, particularly at physiologi- cal ionic strength, the allosteric properties of mammalian brain AChE in vivo have yet to be defined.

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ANDERSEN, R. A., AAROAS, I., GAARE, G., AND FON- NUM, F. (1977). Inhibition of acetylcholinesterase from different species by organophosphoms com- pounds, carbamates and methylsulphonyl fluoride. Gen. Pharmacol. 8,33 l-334.

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HART, G. J., AND O’BRIEN, R. D. (1974). Stopped-flow studies of the inhibition of acetylcholinesterase by or- ganophosphates in the presence of substrate. Pestic. Biochem. Physiol. 4,239-244.

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HORTON, G. L., LOWE, J. R., AND LIESKE, C. N. (1977).

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PETER J. GRAY’

RAYMOND M. DAWSON

Materials Research Laboratories

Defence Science and Technology Organization P.O. Box 50 Ascot Vale VIC 3032

.4ustralia

Received December 15.1986; accepted June 29, I987

’ To whom correspondence should be addressed