Microcalorimetric determination of binding sites of acetylcholinesterase

Biochimica et Biophysica Acta, 745 (1983) 107-110

107

Elsevier

BBA Report

BBA 30046

MICROCALORIMETRIC DETERMINATION OF BINDING SITES OF ACETYLCHOLINESTERASE * Y.T. DAS, H.D. BROWN ** and S.K. CHATTOPADHYAY

Rutgers University, New Jersey Agricultural Experiment Station, Cook College, P.O. Box 231, New Brunswick, NJ 08903 (U.S.A.) (Received July 19th, 1982) (Revised manuscript received March 22nd, 1983)

Key words: Acetylcholinesterase; Binding site; Enzyme inhibition; Microcalorimetry; Ligand binding; (E. electricus)

Acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7), phosphorylated with dichlorvos, showee relatively more reactivity toward the substrate indophenyl acetate than the enzyme that was carbamylated with carbaryl. When the anionic subsite of the phosphorylated or earbamylated enzyme was alkylated with an aziridinium ion, the reaction velocity toward indophenyl acetate increased in the phosphorylated enzyme but not in the carbamylated enzyme. The organophosphate binding site - - which appears to he different from that of carbamate or indophenyl acetate, but probably the same as that of aeetylcholine - - is apparently alkylated in such a way that the modified (phosphorylated) enzyme is better-fit for the binding (and hydrolysis) of indophenyl acetate. The modified conformation presumably results in the release of the phosphoryi group from the esteratic subsite.

Acetylcholinesterase (EC 3.1.1.7) catalyzes the hydrolysis of acetylcholine and other acetic acid esters, as well as certain esters and acyl halides of substituted phosphoric, carbamic and sulfonic acids. In view of the diversity of these molecules, it is generally believed that their binding patterns differ, while the hydrolytic part of the enzymic reaction remains the same. Although the widely used (pesticidal) esters of phosphoric acid and carbamic acid are generally believed to exert their anticholinergic toxic effects essentially in a similar manner, certain critical differences in their bind-

* New Jersey Agricultural Experiment Station Publication No. D-10511-40102-3/1-82. ** To whom correspondence should be addressed. Abbreviations: dichlorvos, phosphoric acid 2,2-dichloroethenyl dimethyl ester; carbaryl, l-naphthalenol methylcarbamate; MCP, 2-chloro-N-(chloroethyl)-N-methyl-2-phenylethylamine. 0167-4838/83/$03.00 © 1983 Elsevier Science Publishers B.V.

ing, phosphorylation/carbamylation and dephosphorylation/decarbamylation remain to be determined. In our present study, we have explored the binding sites of acetylcholinesterase with selected substrates, inhibitors and an alkylating agent for information on the enzyme molecular topography and subsite interrelationships. The microcalorimetric technique provided direct measurement of the enzymic reactions in a single step without the complication imposed by the measurement of coupled (secondary) reactions. Acetylcholinesterase of Electrophorous electricus was purchased from Worthington Biochemical Corporation, Freehold, NJ (activity: 100 units/mg protein), acetylcholine chloride from Sigma Chemical Company, St. Louis, MO, and indophenyl acetate from Eastman Kodak Company, Rochester, NY. Dichlorvos (98% pure) and carbaryl (100~ pure) were generously supplied by Shell Chemical Company, San Ramon, CA, and Union Carbide

108 c1

--~

--> ~ik

/+%

Fig. 1. Chemical structures of M C P (I) and its aziridinium ion (II).

Corporation, Salinas, CA, respectively. 2-ChloroN-(chloroethyl)-N-methyl-2-phenylethylamine (MCP) was a gift from Dr. D.J. Triggle, School of Pharmacy, State University of New York, Buffalo, NY (Fig. 1). Micromolar amounts of dichlorvos and carbaryl were conveniently measured from stock solutions made in acetone and the solvent was subsequently evaporated at 27°C. The enzyme (100 U) in buffer solution (0.5 ml 0.1 M Tris-HCl, p H 8.0) was added to the glass vial containing the inhibitor (4.0 #mol) and incubated at 25°C for 15 rain with constant stirring. The inhibitor molarity (0.8 mM) was approx. 100-fold higher than the known equilibrium (or dissociation) constant of dichlorvos or carbaryl [1,2]. For alkylation of the enzyme, 0.5 ml of 0.4 mM MCP solution in the buffer was added to 0.5 ml of the enzyme solution and incubated at 25°C for 3 h with constant stirring. Non-alkylated enzyme was stirred in a similar manner with 0.5 ml buffer for 3 h. Inhibition of the enzyme was established by the spectrophotometric method of Ellman et al. [3] using acetylthiocholine as the substrate. The phosphorylated enzyme as well as the carbamylated enzyme yielded no measurable activity, indicating a 'complete' inhibition. Reaction heats were measured with a TianCalvet microcalorimeter constructed in our laboratory, similar in design to the instrument described by Evans [4], using a technique which we have earlier standardized for cholinesterase assay [5]. The two reactants (enzyme and substrate) were placed in the two compartments of the calorimeter cuvette. Upon mixing of the reactants inside the calorimeter the resulting reaction heat was translated by the differential thermophiles into a voltage signal. The latter was traced on a chart recorder for the measurement of maximum (peak) height from the initial (base) line and the area

under the curve for a 10-rain period. Since the observed peak height area included non-catalytic and mixing-dilution heats, control reactions were run with buffer and water in lieu of enzyme and substrate, respectively. These values were then subtracted from the enzymic reaction values to obtain the net peak height and area. The net areas were converted into heat units (mcal) using a standard curve established with a series of neutralization reactions (Tris-HC1) of known enthalpy values [6]. The chart recorder tracings of the 10-rain reactions are presented in Fig. 2. The substrate, which was at a sub-saturation level with reference to the untreated enzyme, yielded a thermogram that typified a quick and complete catalytic reaction with a sharp peak and a return to the baseline. The net peak height (cm) of the untreated enzyme was taken as the highest velocity of the reaction and was used as a reference to evaluate the extent of inhibition or activation (Table I). The net area represented the total substrate consumed in a 10min period. In the case of the uninhibited enzyme reaction, it could be seen that the substrate was 'completely' consumed in the 10-min period. Thus, the net area of the untreated enzyme served as a references to evaluate the slow and protracted reactions. It should be noted here that the 'complete' heat in the slow reactions could eventually be realized over a period of time that is proportional to the number of uninhibited active sites of the enzyme, provided that all other conditions are maintained favorable for the complete consumption of the substrate. It could be seen that the phosphorylation or

12 Untreated

A~~kyl....

10

Phosphorylated & Alkylated Phosphorylated Carbamylated6 A l k y l a t e d C.irbamylated Buffer

8 6

£ 2 0 l

2

3

4

5

6

7

8

9

I 10

time (min)

Fig. 2. Microcalofimetfic output of reactions between ind o p b e n ~ a ~ t a ~ and various enzyme preparations. For ~ a c tion dermis, see text and Table I.

109 TABLE I EFFECT OF ALKYLATION (WITH 0.2 Fmol MCP, 0.2 raM) ON THE REACTION BETWEEN PHOSPHORYLATED(WITH 0.4 Fmol DICHLORVOS,0.8 raM) OR CARBAMYLATED(WITH 0.4 Fmol CARBARYL,0.8 mM) ACETYLCHOLINESTERASE (100 UNITS IN 1.0 ml) AND INDOPHENYL ACETATE (4.0 Fmol, 4.0 mM) 1 ml indophenyl acetate solution (4.0 raM) was reacted with 1.0 ml enzyme preparation, making the total volume of the reaction mixture 2.0 mi. Consequently, the individual molarities in the final reaction mixture were: MCP, 0.05 raM; dichlorvosor carbaryl, 0.2 mM; indophenyl acetate, 2.0 mM. Velocityis expressedas net peak height (cm). Heat is expressedas net area under the curve/10 rain converted into mcal using a standard curve established with a series of Tris-HCl neutralization reactions. For calibration procedures, see Berger [6] and O'Farrell et al. [111. Percentagevelocity and heat values are percent of corresponding uninhibited enzyme values, viz., non-alkylated enzyme: velocity= 9.1 cm, heat = 12.96 mcal; alkylated enzyme: velocity= 7.6, heat = 11.08 mcal. (Measurements were limited to 10-rain reaction period.) Difference percent is between alkylated enzyme and non-alkylated enzyme. Inhibition

Phosphorylation Carbamylation

Non-alkylated enzyme Velocity Heat

Alkylated enzyme Velocity Heat

Difference(%) Velocity Heat

cm

%

meal

%

cm

%

mcal

%

2.8 2.3

30.8 25.3

9.5 2.7

73.4 20.8

5.2 2.2

68.4 28.9

10.99 2.79

99.2 25.2

carbamylation of the enzyme reduced the velocity by 70-75%. However, the phosphorylated reaction went to 73% of completion in 10 min, as compared with 21% of the carbamylated enzyme. The effect of alkylation on these inhibited enzymes was evaluated by arriving at the difference in the reaction velocities between the alkylated and non-alkylated enzyme (Table I). The phosphorylated enzyme gained 37.6% activity upon alkylation. By contrast, the carbamylated enzyme showed a negligible chain (3.6%). A l t h o u g h acetylcholinesterase shares its nucleophilic activity of a serine hydroxyl group with many other hydrolases, notably chymotrypsin, its substrate-specificity stems from the still ill-defined binding phenomenon. The large variation in the chemical structure among the substrates and inhibitors that react with the enzyme precluded any attempt to oversimplify or unify the binding patterns. The aziridinium ion of MCP has been known to irreversibly (covalently) alkylate an anionic subsite with the loss of enzyme activity toward acetylcholine [7] but not toward indophenyl acetate [8], the latter binding at a presumably different subsite. As per our spectrophotometric evaluation with acetylthiocholine as the substrate, the phosphorylated, carbamylated or alkylated enzyme was 'completely' inactive. However, when the evaluation was made with indophenyl acetate as the

37.6 3.6

25.8 4.4

substrate in the microcalorimeter using 100 U acetylcholinesterase, the alkylated enzyme showed most of its activity (85.5%), the carbamylated enzyme showed only 20.8% activity, but the phosphorylated enzyme showed unexpectedly high activity (73.4%) (Table I). Since no other predisposing conditions for this level of activity were present in the reaction system, we attributed this to an induced effect of the indophenyl acetate binding interactions with enzyme. Interestingly, only the phosphorylated enzyme, but not the carbamylated enzyme, showed increased velocity upon alkylation. Alkylation was believed to alter the enzyme in its binding [9] and activation energy [10] in favor of indophenyl acetate. Although our present microcalorimetric data do not permit critical evaluation of these aspects, they do reveal a striking effect of alkylation on the reaction velocity of the phosphorylated enzyme compared with that of non-phosphorylated enzyme (Table I). Unfortunately, a direct evaluation of the esteratic activity with a cationic substrate such as acetylthiocholine was not possible, nor was a direct measurement of the released phosphate group obtained. The aziridinium ion appears to have formed a ternary complex with the phosphorylated enzyme that was acceleratory toward the indophenyl acetate hydrolysis. This ternary complex is comparable to the one between the acyl enzyme and 1-naphthyl acetate in the presence of an aromatic

I10

cation (pralidoxime) [10], but, unlike it, is an acceleratory complex. It is, therefore, difficult to attribute the increase in reaction velocity to the ternary complex alone without conceiving of an increase in the number of esteratic subsites. The latter situation implies a partial dephosphorylation of esteratic serine either by an allosteric effect of subsite alkylation or by a ternary complex-induced steric change. We are currently exploring noncatalytic methods to obtain direct evidence for the suspected dephosphorylation. We are thankful to Dr. T.L. Rosenberry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, for many helpful discussions. We are also thankful to Dr. R.D. O'Brien, University of Rochester, Rochester, NY 14627, for a review of the manuscript. This work was supported by State funds, U.S. Hatch Act and National Institutes of Health Grant No. PHSGM22679.

Rderences 1 Eto, M. (1974) Organophosphate pesticides: Organic and biological chemistry, p. 387, Chemical Rubber Company, Cleveland, OH 2 Kuhr, R.J. and Dorough, H.W. (1976) Carbamate Insecticides: Chemistry, Biochemistry and Toxicology, p. 301, Chemical Rubber Company, Cleveland, OH 3 Ellman, G.L., Courtney, K.D., Andres, V. and Featherstone, R.M. (1961) Biochem. Pharmacol. 7, 88-95 4 Evans, W.J. (1969) In Biochemical Microcalorimetry(H.D. Brown, ed.), pp. 257-272, Academic Press, New York 5 Rosenstein, S. and Brown, H.D. (1980) Biochim. Biophys. Acta 629, 195-198 6 Berger, R.L. (1969) In Biochemical Microcalorimetry(H.D. Brown, ed.), pp. 227-233, Academic Press, New York 7 Belleau, B. and Tani, H. (1966) Mol. Pharmacol. 2, 411-422 8 Purdie, J.E. and Mclvor, R.A. (1966) Biochim. Biophys. Acta 128, 590-593 90'Brien, R.D. (1969) Biochem. J. 133, 713-719 10 Rosenberry, T.L. (1975) Adv. Enzymol. Relat. Areas Mol. Biol. 43, 103-218 l l O'Farrell, H.K., Chattopadhyay, S.K. and Brown, H.D. (1977) Clin. Chem. 23, 1853-1856