Eur. J . Biochem. 140, 153-156 (1984) ,Q FEBS 1984

Kinetics for the inhibition of acetylcholinesterase from the electric eel by some organophosphates and carbamates

Ake FORSBERG and Gertrud PUU Division of Experimental Medicine, National Defence Research Institute, Umeg

(Received October 25/December 19, 1983) - EJB 83 1155

The inhibition kinetics for some organophosphates (paraoxon, diisopropylfluorophosphate, sarin, VX, soman and soman isomers) and carbamates (physostigmine, neostigmine, pyridostigmine and carbaryl) in the reaction with acetylcholinesterase from electric eel have been studied. Dissociation constants and rate constants for the irreversible step were determined. The great differences in inhibitory power of the organophosphates were almost entirely due to differences in affinity. A possible correlation between affinity and bonding rate is discussed.

Organophosphates and carbamates inhibit cholinesterases by binding covalently to a serine residue in the active site of the enzyme. The inhibitory power of an anticholinesterase is usually expressed as I,,, i.e. the concentration of the inhibitor giving 50% inhibition under defined conditions, or as the bimolecular rate constant, k,, for the reaction

ChEOH + IX ChEOI + HX (1)

where ChEOH represents the active enzyme and IX the inhibitor, with the leaving group X. Aldridge suggested how- ever, more than 30 years ago [I], that the inhibition proceeds in two steps, with the formation of a reversible Michaelis complex preceding the irreversible step.

ChEOH + I X A C h E O H . IX % ChEOI + HX. (2)

The inhibition can thus be characterized by two parameters, the dissociation constant Kd (i.e. k - l /kl) and the unimolecular bonding rate constant, k,. The ratio between these two constants, k, /Kd, gives an overall rate of inhibition, ki, which should be called the bimolecular reaction constant [2]. Compared to the immense literature on ki and Z5, values for various cholinesterase inhibitors, relatively few substances have been subjected to Kd and k , analysis. This may be due to experimental difficulties, mainly arising from the desirability of using inhibitor concentrations close to Kd values. The experi- mental difficulties become less pronounced when using a method developed by Hart and O'Brien 131. Inhibitor con- centrations can be higher, as the inhibition reaction takes place simultaneously with the substrate reaction. By using a chromo- genic substrate the progressive inhibition can be continuously

L ~ ,

Abbreviations. Dip-F, diisopropylfluorophosphate; Paraoxon, diethyl p-nitrophenylphosphate; sarin, isopropyl methylfluorophos- phonate; soman, 1,2,2-trimethylpropyI methylfluorophosphonate; VX, ethyl S-diisopropylaminoethyl methylthiophosphonate; Carb- aryl, I-naphthyl N-methylcarbamate; Neostigmine, (m-hydroxy- pheny1)-trimethylammonium methylsulfate dimethylcarbamate; Pyridostigmine, 3-hydroxy-1 -methylpyridinium bromide dimethylcar- bamate; Physostigmine, (3aS-cis)-1,2,3,3a,8,8a-hexahydro-l,3a,8- trimethylpyrrolo[2,3-b]indol-S-ol methylcarbamate.

Enzyme. Acetylcholinesterase (EC 3.1.1.7).

registered photometrically. The method has been further developed using stopped-flow technique [4, 51, which is prefer- able when studying substances with high k , values.

We have studied the inhibition kinetics for some potent organophosphates, e.g. nerve agents, and carbamates, using a fast-responding and sensitive conventional photometer. For most of the substances we were able to use inhibitor con- centrations approaching the Kd values found.

MATERIALS AND METHODS

Acetylcholinesterase (EC 3.1.1.7) from electric eel, type V-S, p-nitrophenyl acetate, neostigmine methyl sulfate and eserine salicylate salt were all purchased from Sigma Chemical Co. Pyridostigmine bromide was a gift from Roche-Produkter AB (Skarholmen, Sweden). Carbaryl and all organophos- phorus compounds were synthesized at the Chemistry Department (National Defence Research Institute, UmeP, Sweden).

A stock solution of enzyme in 0.067 M sodium phosphate buffer pH 6.9 was kept frozen. Dilution was made freshly each day to give 200 units m1-I.

The substrate, p-nitrophenyl acetate, was dissolved in absolute ethanol, freshly each day. Immediately before a set of experiments, dilution was made with phosphate buffer to give the appropriate concentration. i.e. for the inhibition experi- ments 2.5mM. Ethanol concentration in the enzyme assay medium was 1 .O % which had no influence on enzyme activity.

All the carbamates and paraoxon were dissolved directly in the buffer mentioned above. Stock solutions of sarin, soman, VX and Dip-F were prepared in isopropanol, freshly each day, and diluted with buffer immediately before use. The con- centration of isopropanol in the assay medium never exceeded 0.1 and had no effect on the activity of the enzyme. The amount of active sites in the enzyme preparation was de- termined, by soman titration essentially according to Schoene 161 to be 0.4pmol/enzyme unit.

For all photometric measurements, a Perkin Elmer model 557 spectrophotometer was used. The photometer was equip- ped with a mixing plunger accessory, with which enzyme solution was added to the cuvette. The formation of p-nitro- phenolate was registered at 402 nm.

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All experiments were performed at 22 “C in 0.067 M sodium phosphate buffer pHh.9 in a total volume of 2.50ml. The enzymatic hydrolysis of p-nitrophenyl acetate was started by addition of 25 p1 enzyme (5 units). Thus, the concentration of active sites in the cuvette was 0.8 nM.

Before each set of inhibition experiments, the K, was determined by using between six and eight different con- centrations of p-nitrophenyl acetate (0.75 - 2 mM). To correct for spontaneous hydrolysis of the substrate, reference cuvettes with the same concentrations of substrate as in corresponding sample cuvettes were used. K,,, was calculated by the Eadie- Hofstee plot. For the calculation of inhibition constants reported here, K,, values in the range 2.6 - 2.75 mM were used.

A substrate concentration of I m M was chosen for all inhibition experiments. Buffer, substrate and inhibitor of various concentrations were added to six, seven or eight cuvettes. Reference cuvettes, with the same contents as the corresponding sample cuvette, were also prepared, as well as cuvettes containing no inhibitor (100 ‘i: activity, vc). A sample cuvette and corresponding reference cuvette was transferred to the photometer, registration was started, enzyme was added and the sample was thoroughly mixed. By using reference cuvettes, a correction was made directly for non-enzymatic reactions. To correct for possible residual enzyme activity [5, 71, i.e. the enzymes shows some slight activity even after prolonged incubation with the inhibitor, the course of the reaction for the sample with the highest inhibitor concentration was followed during several minutes. The substrate was regarded as giving residual activity if a small. monotonous increase in absorbance could still be noticed after 5 min.

To each progressive inhibition curve, tangents were drawn, freehand, a t each I0 s for the slower courses, at each 5 s for the faster. The slopes of the tangents were calculated, residual activity (if there was any) was substracted and the (corrected) slopes were plotted in a semilogarithmic diagram against time (primary plot). For all the inhibitors and concentrations used, the resulting plots were linear. The slopes (Ah v / A t ) were determined by the least-squares method.

The slopes thus obtained were used for further analysis, applying the double-reciprocal method [3], which takes advan- tage of the relationship

1 + - 1In v h , [IX] (I-a) k , 1 ’ t - - Kd ~ x

where [IX] is inhibitor concentration and a stands for [S]/ ( K , + [S]); where [S] is substrate concentration (1 mM in all experiments) and K, is the Michaelis constant, determined in a separate experiment. Thus, from a plot of At/Aln v against I/[IX] (1-a) (secondary plot), Kd is determined as the reciprocal value of the intercept on the abscissa and k, as the reciprocal value of the intercept on the ordinate.

The analysis assumes that the initial binding step is an equilibrium, i.c. k, is much smaller than k - This can not be shown directly. However, k , would have to be extremely small for the equilibrium assumption not to be valid.

RESULTS

The progressive inhibition process is illustrated in Fig. 1, which shows the inhibition ofacetylcholinesterase by the nerve agent sarin in three different concentrations. At higher concen- trations of such potent anti-cholinesterases, the progressive curve could be followed for only 25 - 30 s and the changes in

/

t E

AAz0.0025 t

t E

t E t

E

- 10s

Fig. 1. The progressive decline in esteratic activity in ilie presence of surin. To a cuvette containing 0.067M sodium phosphate buffer pH6.9, 1 mM substrate (p-nitrophenyl acetate) and sarin (0.1, 0.5 or 1 .0 pM), 5 units of electric eel acetylcholinesterase was added at t = 0 (see arrows labelled E). The formation of p-nitrophenolate was registered as absorbance at 402 nm. Note the increasing amplification at higher sarin concentration. v, denotes activity in absence of inhibitor

absorbance were small. They could, however, be registered and analyzed without difficulties. With one exception, the highest concentration used for each inhibitor was close to or higher than the observed dissociation constant.

For sarin, the lowest concentration used was 0.1 pM, and for soman and VX even lower concentrations were used. The ratio between inhibitor and enzyme concentrations could consequently be considered as UnfdVoUrabk, at least at the beginning of the reaction. However, no deviations from linearity could be detected when plotting In 1’ versus t (primary plots), suggesting that the reaction followed pseudo-first-order kinetics.

In Tables 1 and 2 we have summarized our results. For the organophosphates (Table 1) the great differences in overall inhibitory power were almost entirely due to differences in affinity. For example, a comparison between the substances with lowest and highest Kd, soman and Dip-F, shows that the affinity of soman was about 50000-times greater than the affinity of Dip-F. The differences in phosphorylation rate were far smaller. VX, which has a thiocholine-resembling structure as leaving group, had the fastest phosphorylation. This high bonding rate is in agreement with the finding that for VX it was not possible to use concentrations approaching the observed Kd value (Table 1).

We paid special attention to soman, which contains (apart from an asymmetric phosphorus in common with sarin, for example) an asymmetric carbon in the pinacolyl moiety. There are thus four stereoisomers of soman, &R,, &Sp, S,R, and S,S,. We performed inhibition kinetic studies on soman, resolved as regards the asymmetric a-carbon, i.e. on &- and S,-soman. These are thus mixtures of two diastereomers, &R,/&S, and S,R,/S,S,, respectively, but the contribution of the S, forms can be ignored as they are about 1000 times less effective anticholinesterases than the R, forms (81. As shown in Table 1, the S, form is the most potent inhibitor, having a lower Kd than &, but the differences were small in the studied system.

All carbamates studied (Table 2) contain a ring structure. Carbaryl and eserine are methylcarbamates, neostigmine and

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Table 1. Kinetic constants f o r the inhibition of eel acetylcholinesterase by some organophosphorus compounds Inhibition proceeded in the presence of 1 mMp-nitrophenyl acetate as substrate at 22 "C in 0.067 M sodium phosphate buffer pH 6.9. Kd and k , were determined by the double-reciprocal method and linear regression. Standard errors are given in parentheses

Inhibitor Concentration k2 range

ki

Dip-F (diisopropylfluoro- phosphate)

Paraoxon (diethyl p-nitro- phenylphosphate)

VX (ethyl S-diisopropyl amino- ethyl me thy1 thi oph osp honate)

Sarin (isopropyl methylfluoro- phosphonate)

Soman (1,2,2-trimethylpropyl methylfluor ophosphonate)

&-soman &-soman

s - 1

0.1-2 mM 0.17 (0.01)

20- 125 PM 0.33 (0.04)

50- 500 nM 0.91 (0.32)

0.1 - 1 PM 0.36 (o.ioj

40 - 200 nM 0.57 (0.15) 40 - 250 nM 0.56 (0.17) 40- 200 nM 0.57 (0.21)

M

1.1 10-3 (0.1)

1.7 x (0.3)

2.1 x (0.5)

1.4 x (0.3)

6.1 x lo-' (1.7) 8.2 x lo-' (2.5) 5.4 10-7 (1.7)

M-ls-l

1.58 x 10'

1.91 103

4.24 105

2.59 105

9.30 105 6.80 105

10.54 x lo5

Table 2. Kinetic constants .for the inhibition of' eel acetylcholinesterase by some carbamateh Details are the same as for Table 1

Inhibitor Concentration k2 Kd ki range

S - 1 M M-Is-l P M Carbaryl (1-naphthyl N-methylcarbamate) 20 - 300 0.33 (0.05) 5.0 x (0.7) 6.54 x 10, Pyridostigmine (3-hydroxy-1-methyl-

pyridinium bromide dimethylcarbamate) 40 - 400 0.057 (0.002) 6.8 x (0.6) 8.43 x 10' Physostigmine {(3aS-cis)-1,2,3,3a,8,8a- hexahydro-l,3a,S-trimethylpyrrolo [2,3-b]indoI-5-ol methylcarbamate) 1.5-40 0.32 (0.02) 1.7 10-5 (0.2) 1.85 104

ammonium methylsulfate dimethylcarbamate] 0.6 - 5 0.57 (0.18) 1.4 10-5 (0.3) 4.09 104 Neostigmine [(m-hydroxypheny1)trimethyl-

pyridostigmine dimethylcarbamates. The two last-mentioned contain quarternary nitrogen, which could be assumed to facilitate the association to the active site by binding to the anionic binding site of the enzyme. Neostigmine was also found to have the lowest Kd, but the affinity of eserine, containing a protonised tertiary nitrogen, was almost the same. Pyrido- stigmine had the lowest carbamylation rate of the studied substances.

DISCUSSION

Dip-F, sarin and soman are closely related compounds but still very different in inhibitory power. From our data it can be noted that the main difference is in affinity for the enzyme, but phosphorylation rate increases also, in the same sequence. Is there a correlation between Kd and k,, at least for closely related inhibitors? We tested this hypothesis by putting to- gether all available inhibition constants for organophosphates and eel acetylcholinesterase, obtained by Hart and O'Brien's method, and performing a linear regression of -log Kd vs k,. The analysis thus included data for the seven compounds reported in this work, Amiton and paraoxon from the work by Horton et al. [5] and three organophosphinates reported by Lieske et al. [7]. A correlation coefficient of I = 0.79 was obtained for these 12 inhibitors.

For the carbamates studied by us, there is no obvious relationship between Kd and k,. For a series ofneostigmine and neostigmine-related carbamates, reported by Iverson and Main [9], we obtained a correlation coefficient of r = 0.81 when plotting -log Kd vs k,. The dissociation constant, reflecting affinity to the active site of the enzyme, and the bonding rate constant are usually regarded as two parameters independent of each other. The rate constant for phosphorylation and carbamylation should largely be determined by the elec- trophilicity of the phosphorus and the carbonyl carbon, respectively, but also other factors, e.g. steric ones, could have an influence. The affinity is obviously influenced by several factors, for example size, three-dimensional structure, presence of groups which easily bind noncovalently to groups in or close to the active site etc. A high affinity means that the inhibitor fits very well into the active-site cleft of the enzyme. A consequence of such a good fit could be an orientation in space such that the covalent bonding to the serine residue is facilitated. Thus there might be a correlation between dissociation and bonding rate constants, at least for compounds with closely related structures.

REFERENCES

1. Aldridge, W. N. (1950) Biochein. J. 46, 451 -460. 2. Main, A. R. (1964) Science (Wash. DC) 144, 992-993.

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3. Hart, G. J . & O’Hrien, R. D. (1973) Biochemistry 12, 2940-2945. 4. Hart, G. J. & O’Brien, R. D. (1974) Pestic. Biochem. Physiol. 4,

5. Horton, G. L., Lowe, J . R. & Lieske, C. N. (1977) A i d . Biochem.

6. Schoene, K. (1971) Biochem. Pharmucol. 20, 2527-2529.

7. Lieske, C. N., Clark, J. H., Meyer, H. G., Lawson, M. A., Lowe, J . R., Blumbergs, P. & Priest, M. A. (1982) Pestic. Biochem. Physiol. 17, 142- 148.

8. Keijer, J . H. & Wolring, G. Z. (1969) Biochim. Biophys. Actu 185,

9. Iverson, F. & Main, A. R. (1969) Biochemistry 8, 1889-1895.

239 - 244.

78, 21 3 - 228. 465 ~ 468.

A. Forsberg and C . Puu, Sektion 431, Fdrsvarets Forskningsanstalt, S-901-82 Umea, Sweden