Silicon compounds as substrates and inhibitors of acetylcholinesterase

Biochimica et Biophysica Acta. 791 (1984)278-280

278

Elsevier

BBA Report BBA 30085

SILICON COMPOUNDS AS SUBSTRATES AND INHIBITORS OF ACETYLCHOLINESTERASE AUP.ICA ABERMAN a, DINA SEGAL a, YECHIEL SHALITIN a,. and A R I E L . G U T M A N b

a Departments of Biology and b Chemistry, Technion-lsrael Institute of Technology. Haifa 32000 (Israel) (Received June 13th, 1984)

Key words: Acetylcholinesterase; Competitive inhibitor; Silicon substrate analogue; (Electric eel)

Several trimethylsilyl derivatives were found to be ligands of acetyicholinesterase (acetyicholine acetylhydroiase, EC 3.1.1.7): trimethylsilylethyl acetate (III) and trimethylsHylmethyi acetate (V) are substrates of the enzyme, whereas trimethylsilylethanol (VIII) is a competitive inhibitor. The silicon compounds have kinetic parameters similar to those of their carbon analogues, except for trimethylsilylmethyl acetate, which is a substrate of acetylcholinesterase, whereas its carbon analogue is not susceptible to enzymic hydrolysis.

Acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) has been the subject of intensive studies in the last 4 decades. As acetylcholinesterase plays a central role in transmission of nervous signals, the mechanism of action of the enzyme has been thoroughly investigated and numerous compounds have been examined as substrates or inhibitors of the enzyme, many of which have found use in research, pharmacology and pest control. Acetylcholine, the cationic natural substrate, is an excellent substrate of acetylcholinesterase, but other acetate esters, especially those derived from acidic alcohols, are also very good substrates [1-8]. Neutral analogues of acetylcholine, in which a carbon atom replaced the nitrogen atom of acetylcholine, are also substrates, Thus, 3,3-dimethylbutyl acetate, which is isosteric with acetylcholine, is a good substrate [4,5]. In recent years, organosilicon compounds have attracted considerable attention, and some silicon derivatives have been found to be bioactive and to mimic the pharmacological behaviour of their

* To whom correspondence should be addressed. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

carbon analogues [9]. It was of interest to examine whether silicon analogues of acetylcholine and choline are recognized by the enzyme and can serve as ligands. Reagents used in the study were of analytical grade. Acetylcholinesterase from electric eel was purchased from Worthington or Sigma. Acetylcholine chloride was from Fluka. Trimethylsilylmethyl acetate, trimethylsilylethanol, choline, 3,3dimethylbutanol were from Sigma and Aldrich. 3,3-Dimethylbutyl acetate and 2,2-dimethylpropyl acetate (neopentyl acetate) were prepared by acetylation of the corresponding alcohols according to literature procedures [10]. Trimethylsilylethyl acetate was prepared by an adaptation of a recently published method for acylation of sterically hindered alcohols [11]: acetic anhydride (8 mmol) was added to a solution of 2-trimethylsilylethanol (5 mmol) and 4-dimethylaminopyridine (10 mmol) in dichloromethane (10 ml). After standing at room temperature for 20 min, water (30 ml) and diethyl ether (50 ml) were added, this being followed by extraction with 0.5 M HC1 (3 × 20 ml) and saturated NaHCO 3 solution (3 × 20 ml). The diethyl ether layer was washed with water (20 ml) and dried over Na2SO 4 and most of the diethyl

279 ether was removed by distillation at atmospheric pressure. The residue was submitted to bulb-tobulb distillation to give 2-trimethylsilylethyl acetate in 80% yield, b.p. 97-100°C at 75 mmHg. (lit. 5 2 - 5 5 ° C at 22 m m H g [12]). N M R spectrum was in excellent agreement with the product identification. The enzyme-catalyzed hydrolysis of the esters was followed pH-statically. Non-cationic compounds were dissolved in dioxane and an aliquot was added to the pH-stat vessel. The final concentration of dioxane was 1%. The reaction solution contained 0.1 M NaC1, 0.02 M MgCI 2 and enzyme. Substrate concentrations were generally in the 0.5-10 m M range. The reaction was run at p H 8, 25°C, and 10 m M N a O H was the titrant. Lineweaver-Burk double-reciprocal plots yielded straight lines, from which the kinetic parameters kcat and K m were calculated. In order to determine the inhibition constant, inhibitor in different concentrations was added to solutions containing constant concentration of acetylcholine. K i was calculated from a Dixon plot [13] of 1 / v vs. [I]. Acetylcholinesterase concentration was determined by active-site titration with phenothiazine-10carbonylchloride [6]. Several silicon compounds were studied as AChE substrates, and choline analogues were tested as inhibitors. A representative Lineweaver-Burk plot of enzymic hydrolysis of trimethylsilylethyl acetate is given in Fig. 1. Fig. 2 is Dixon plot of the inhibition of acetylcholinesterase by trimethylsilylethanol, an analogue of choline. Such lines are characteristic

4

I 0.2

I I I 0.4 0.6 0.8 I/[S] ,raM -=

I I

I 1.2

Fig. 1. Double-reciprocalplot of acetylcholinesterase-catalyzed hydrolysis of trimethylsilylethylacetate, pH 8.0, 25°C. o is the reaction rate in rain-1 (tool substrate hydrolyzed per mol enzyme per min) for the specified substrate concentration. Reaction solution contained 0.1 M NaCI, 0.02 M MgCI2 and 1% dioxane. Enzyme concentration 0.6 nM. of competitive or mixed inhibition. However, analysis of the graph indicates that the inhibition in this case is competitive. The kinetic parameters found for analogue substrates and inhibitors of acetylchofinesterase are listed in Table I. It is seen that substitution of silicon for nitrogen or carbon results in an effective substrate or inhibitor, with similar values of kinetics parameters. The surprising finding is that trimethylsilylmethyl acetate (V) is a substrate of acetylcholinesterase, whereas its carbon analogue 2,2-dimethylpropyl acetate (neopentyl acetate) (IV) is not hydrolyzed by the enzyme. Even when acetylcholinesterase concentration with IV was 10-fold higher than that used with V, the former failed to show enzyme-catalyzed hydrolysis.

TABLE I KINETIC PARAMETERS OF SILICON SUBSTRATES AND INHIBITORS OF ACETYLCHOLINESTERASEAND THEIR ANALOGUES n.d., not detectable. Substrate or inhibitor I acetylcholine II 3,3-dimethylbutylacetate III 2-trimethylsilylethylacetate IV 2,2-dimethylpropylacetate V 2-trimethylsilylmethylacetate VI choline Vll 3,3-dimethylbutanol VIII 2-trimethylsilylethanol

(CH3)3N+ CH2CH2OCOCH3 (CH 3) 3CCH 2CH2OCOCH3 (CH3)3SiCH2CH 20COCH 3 (CH3)3CCH2OCOCH3 (CH3)3SiCH 2OCOCH3

(CH3)3N+ CH2CH2OH (CH3)3CCH2CH2OH (CH3)3SiCH2CH2OH

kcat (rain- 1)( x 10- 5)

Km(mM)

9.6±0.2 4.1 ± 0.3 3.1 ± 0.3 n.d. 1.8 ± 0.2

0.5±0.1 5 ±0.4 1.3±0.2

Ki (mM)

7.5±0.5 1 ±0.1

0.8±0.1 3.8±0.5

280

2

v. I

I

I

I

I

-5

5

I0

15

I'll ,ram Fig. 2. Dixon plot of the inhibition of acetylcholinesterasecatalyzed hydrolysis of acetylcholine (1.5 mM and 0.3 mM) by trimethylsilylethanol (pH 8.0), 25°C. v is the reaction rate and v0 is the rate of the reference reaction 1.5 mM acetylcholine in absence of inhibitor.

Substitution of carbon by silicon does not seriously affect the reactivity of the compound. Thus, trimethylsilylalkyl acetates undergo alkaline and acidic hydrolysis at a rate which is 70% the rate of their carbon analogues [14,15]. On this basis, it is expected that IV would display higher reactivity with the enzyme than V. It seems therefore that the susceptibility of V and the resistance of IV to acetylcholinesterase do not originate from chemical reactivity, but rather from other reasons, probably steric fitness. The trimethylsilyl group is larger than its carbon or nitrogen analogues. The bond length of N-C is 1.48 .A; that of C-C is 1.54 .~ and that of Si-C is 1.86 A [16]. It is plausible that the bulky trimethylsilyl group of V can fit itself and accommodate into the binding site of the enzyme, which has a hydrophobic nature [1,5], whereas the carbon analogue IV which is smaller by 0.64 A does not fit. That V binds to the enzyme in the normal binding site was supported by addition of tetramethylammonium chloride to the reaction solution. The salt was found to be a competitive inhibitor of the enzyme reaction, with Ki-l.1 mM. This value was also found for the inhibition of the reaction of acetylcholinesterase with acetylcholine

and other substrates with the above ammonium salt [3,17]. Choline and its carbon and silicon analogue are all competitive inhibitors of acetylcholinesterase, having K i in the millimolar range. However, the silicon analogue binds somewhat less tightly to acetylcholinesterase than do choline and its carbon analogue, and its standard free energy of binding is less favorable by 800 cal/mol than that of choline. The variety in the affinities of choline and its analogues to acetylcholinesterase is probably due to differences in the sizes of the ligands. This study was supported by the Technion Vice President Research Fund. References 1 Froede, H.C. and Wilson, I.B. (1971) The Enzymes, 3rd Edn. (Boyer, P.D., ed.), Voi. 5, 87-114, Academic Press, New York 2 Rosenberry, T.L. (1975) Adv. Enzymol. 43, 103-218 3 Krupka, R.M. (1966) Biochemistry 5, 1988-1998 4 J~Lrv, J., Kesvetera, T. and Aaviksaar, A. (1976) Eur. J. Biochem. 67, 315-322 5 Hasan, F.B., Cohen, S.G. and Cohen, J.B. (1980) J. Biol. Chem. 255, 3898-3904 6 Naveh, M., Segal, D., Bernstein, Z. and Shalitin, Y. (1981) FEBS Lett. 134, 53-56 7 Bracha, P. and O'Brien, R.D. (1968) Biochemistry 7, 1545-1554 8 Bracha, P. and O'Brien, R.D. (1968) Biochemistry 7, 1555-1559 9 Tacke, R. and Wannaget, U. (1979) Topics Curr. Chem. 84, 1-75 10 Soderquist, J.A. and Thompson, K.L. (1978) J. Organometal. Chem. 159, 237-249 11 H~fle, G. and Steglich, W. (1972) Synthesis 619-621 12 Eaborn, C., Mahmoud, F.M.S. and Taylor, R. (1982) J. Chem. Soc. Perkin Trans. II, 1313-1319 13 Dixon, M. (1953) Biochem. J. 55, 170-171 14 Pola, J. and Chralovsky, V. (1974) Collect. Czech. Chem. Commun. 39, 2247-2252 15 Pola, J. and Chralovsky, V. (1974) Collect. Czech. Chem. Commun. 39, 2637-2640 16 Weast, R.C. (ed.) Handbook of Chemistry and Physics (1982) p. F-180, CRC Press, Boca Raton, FL 17 Wilson, I.B. and Alexander, J. (1962) J. Biol. Chem. 237, 1323-1326