ISSN 1607-6729, Doklady Biochemistry and Biophysics, 2006, Vol. 408, pp. 192–195. © Pleiades Publishing, Inc., 2006 Original Russian Text © E.V. Rozengart, N.E. Basova, 2006, published in Doklady Akademii Nauk, 2006, Vol. 408, No. 6, pp. 831–834.
BIOCHEMISTRY, BIOPHYSICS, AND MOLECULAR BIOLOGY
Sulfonium and Ammonium Ligands of the Active Site of Cholinesterases E. V. Rozengart and N. E. Basova Presented by Academician V.L. Sviderskii December 14, 2005 Received December 22, 2005
DOI: 10.1134/S1607672906030227
Cholinesterases are a unique enzyme family in terms of the number and structural diversity of specific effectors (substrates and reversible and irreversible inhibitors) [1, 2]. Although all these thousands of compounds are ligands of cholinesterase active center, they substantially differ with respect to both the site of sorption on the catalytic surface of cholinesterase and the mechanism of sorption process. Substrates have the most stringent requirements for the “seat” that should provide a strictly determined approach to the catalytic triad of the active center of the enzyme, so that the hydroxyl of serine could be acetylated [3, 4]. In the case of irreversible anticholinesterase action of organophosphorous inhibitors (OPIs), these requirements are also very high, because the result of sorption of these quasisubstrates is the formation of a phosphorylated adduct, which implies another (compared to substrates) angle of attack of the hydroxyl of serine in the catalytic triad of the active center of cholinesterase [3, 4]. Finally, quite a different pattern is observed in the case of reversible onium inhibitors, for which the peripheral area of the catalytic surface of cholinesterase may be crucial [1, 2]. The only natural substrate of cholinesterase is acetylcholine, the molecule of which, besides a reactive ester group, contains a “sorptionally important” group in the form of hydrophobic peralkylated ammonium cation. A multiyear structure–kinetics analysis of the mechanism of cholinesterase catalysis has been performed in terms of the structure–activity relationship using the entire set of available cholinesterase effectors whose structures varied considerably [1, 2]. The nature of the onium atom of the hydrophobic cationic group of ligands of the active center of cholinesterases has been studied to a lesser extent. To study the mechanism of cholinesterase reactions, the ammonium atom in the effector onium moiety was substituted in modeling Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, pr. Morisa Toreza 44, St. Petersburg, 194223 Russia
experiments with the sulfonium atom; this was first performed in the molecules of irreversible OPIs [5, 6] and then in the molecules of substrates [7, 8] and reversible onium inhibitors [9, 10]. In all cases, the effect of such substitutions was purely quantitative: the pattern of interaction of all sulfonium ligands with cholinesterase was similar to that of the ammonium analogues, though these ligands differed in the values of kinetic parameters. This was the first study to combine and analyze in detail our own and published data on comparison of the efficiency of sulfonium and ammonium substrates, irreversible OPIs, and reversible inhibitors of cholinesterase. Commercially available human erythrocyte acetylcholinesterase (EC 3.1.1.7) and blood serum butyrylcholinesterase horse blood serum cholinesterase (EC 3.1.1.8) with a specific activity of 1.2 and 9.6 units, respectively (Perm Research Institute of Vaccines and Sera), were used as sources of reference enzymes. Ammonium (compounds 1–4) and sulfonium (compounds 5–8) acetate derivatives were used as substrates (For formulas, see Table 1) [7, 11]. The rate of enzymatic hydrolysis of substrates was determined by potentiometric titration at 25°ë and pH 7.5 [2]. The irreversible sulfonium OPIs (see formulas in Table 2, compounds 9 and 11) were synthesized at the Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences [2]. The data on the ammonium OPIs (see formulas in Table 2, compounds 10 and 12) were extracted from [12]. The anticholinesterase activity of OPIs was estimated in the reactions with cholinesterase in the absence of substrate and characterized by the bimolecular constant kII (M–1 min–1) [2]. The data characterizing the efficiency of ammonium OPIs in the form of negative logarithm of the OPI concentration that causes 50% inhibition, presented in [12], were recalculated by the formula for the bimolecular constant of interaction of OPI with cholinesterase: kII = (2.3/t[I] log ( v0/vt), where I = I50; v0/vt = 2 (provided
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Table 1. Kinetic analysis of hydrolysis of ammonium and sulfonium acetates with the general formula CH3C(O)O(CH2)n − X catalyzed by acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) X=N+(CH3)3 · I–
Compound no.
n
1 2 3 4
1 2 3 4
AChE
BChE
log kII
vrel
log kII
log kII
– 7.70 – –
– 100 70 10
3.20 5.87 3.40 2.35
– 7.00 – –
X=S+(CH3)C2H5 · CH3SO4
Compound no.
n
5 6 7 8
1 2 3 4
AChE
BChE
log kII
log kII
kII, rel
– 7.00 – –
4.15 4.67 3.80 3.30
2 100 13 4
Note: kII is the bimolecular rate constant of enzymatic hydrolysis of acetates: kII = ac /KM, where ac is the activity of the catalytic center of the enzyme (s–1) and KM is the Michaelis constant. Vrel is the relative value of the rate of enzymatic hydrolysis of acetates.
Table 2. Parameters of anticholinesterase effectiveness of sulfonium and ammonium organophosphorous inhibitors [2, 12] Compound no. 9 10 11 12
kII , M–1 min–1
pI50
Inhibitor
AChE
– C2H5O(CH3)P(O)SC2H4S+(CH3)C2H5 · CH3 SO 4 C2H5O(CH3)P(O)SC2H4N+(CH3)3 · I– – (C2H5O)2P(O)SC2H4S+(CH3)C2H5 · CH3 SO 4 (C2H5O)2P(O)SC2H4N+(CH3)3 · I–
– 9.4 – 7.9
BChE
AChE
BChE
– 7.9 – 8.0
1.3 × 9.0 × 108* 6.8 × 106 2.7 × 107*
2.1 × 106 2.7 × 107* 2.9 × 107 3.5 × 107*
108
Note: Designations: pI50 , the negative logarithm of the OPI concentration that causes 50% inhibition of cholinesterase; kII, the bimolecular rate constant of interaction of OPI with cholinesterase; k *II , the calculated values of the bimolecular rate constant (see text).
50% inhibition of cholinesterase); t is the duration of incubation of OPI with cholinesterase (2 min for highly active OPI) [2]. Table 2 summarizes the calculated values of the bimolecular rate constant ( k *II ). We also studied the sulfonium compounds (Table 3, compounds 13–17) synthesized at the Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan [9], their commercially available ammonium analogues (compounds 18–21; Sigma, United States), and tetrabutylphosphonium bromide (compound 22) kindly provided by Skvortsov [13]. All compounds studied (Table 3) proved to be reversible inhibitors of the studied cholinesterases, because (1) their effectiveness did not depend on the duration of incubation with enzymes and (2) their inhibitory effect markedly decreased upon dilution of reaction mixture [1, 2]. The effectiveness of reversible inhibitors was assessed by the aggregate inhibition constant K i , which in the case of mixed inhibition type is related to its competitive (Ki) and uncom1 1 1 petitive ( K 'i ) components by equation ----- = ----- + ----- . K i K i K 'i The inhibitory constants (expressed as the mean values of five to six measurements) were calculated graphically [2]. The inhibition type was determined from the proportion between pKi and p K 'i . Inhibition was DOKLADY BIOCHEMISTRY AND BIOPHYSICS
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regarded competitive (C) if p K 'i → 0, mixed (M) if pKi > p K 'i , noncompetitive (N) if pKi = p K 'i , and uncompetitive (U) if pKi → 0 [2]. Table 1 shows two unique series of onium acetates with different distance between the ammonium and sulfonium groups and an ester bond. Unfortunately, these compounds have been studied in sufficient detail only as substrates of butyrylcholinesterase [7]. In this connection, two things are worth mentioning. First, the rate of enzymatic hydrolysis strongly depended on the acetate structure: in the ammonium and sulfonium series, a pronounced maximum was observed at n = 2 (in the ammonium series, compound 2, acetylcholine (Table 1)). Second, the proportion of kII between series was different. For example, out of the most active substrates, acetylcholine (compound 2; Table 1) was 15 times more effective than the corresponding sulfonium acetate (compound 6), whereas ammonium substrates, such as compounds 1.1 and 1.3, were weaker by a factor of 10 than their sulfonium analogues (compounds 5 and 8). As seen in Table 1, in the case of acetylcholinesterase, acetylcholine (compound 2) was hydrolyzed five times more rapidly than the sulfonium derivative (compound 6). In this study, we for the first time compared the values of the anticholinesterase effect of two pairs of sulfonium and ammonium OPIs—the derivatives of 2006
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Table 3. Parameters of the anticholinesterase efficiency of onium inhibitors Compound no. 13 14 15 16 17 18 19 20 21 22
AChE Inhibitor –
S+(CH3)3 · CH3 SO 4 – S+(C2H5)2CH3 · CH3 SO 4 – S+(C3H7)2CH3 · CH3 SO 4 – S+(C4H9)2CH3 · CH3 SO 4 – S+(C5H11)2CH3 · CH3 SO 4 N+(CH3)4 · I– N+(C2H5)4 · I– N+(C3H7)4 · Br– N+(C4H9)4 · Br– P+(C4H9)4 · Br–
BChE
pK i
pKi
pK 'i
IT
pK i
pKi
pK 'i
IT
2.43 3.57 3.45 3.83 4.14 3.50 3.40 4.48 4.73 3.64
2.39 3.38 2.85 3.71 4.03 3.32 3.26 4.35 4.55 3.32
1.32 3.11 3.33 3.20 3.47 3.00 2.81 4.00 4.27 3.35
M N N M M M M M M N
2.06 2.73 3.21 3.44 4.08 2.45 2.72 3.37 3.41 3.29
1.98 2.73 2.98 3.44 4.08 2.45 2.72 3.37 3.41 3.29
1.28 – 2.82 – – – – – 2.90 –
M C N C C C C C M C
Note: pK i , pKi , and pK i' are negative logarithms of the aggregate inhibition constant and its competitive and noncompetitive components, respectively. Other designations: IT, Inhibition type; C, competitive; M, mixed; N, noncompetitive.
ethoxymethylphosphonic acid (compounds 9 and 10) and diethoxyphosphoric acids (compounds 11 and 12) (Table 2). As mentioned above, we recalculated pI50 for kII for Tammelin’s ammonium OPIs [12], which allowed us to compare their efficiency correctly. It appeared that the ammonium OPIs (compounds 10 and 12) are more effective than their sulfonium analogues (4–7 times in the case of acetylcholinesterase and 10 times in the case of butyrylcholinesterase). Note that the inhibitory effect of ethoxymethylphosphonium OPIs (compounds 9 and 10) with respect to acetylcholinesterase was stronger by an order of magnitude than the inhibitory effect of diethoxyphosphoric OPIs (compounds 11 and 12). In the case of butyrylcholinesterase, an inverted dependence for sulfonium OPIs (compounds 9 and 11) was observed, which agrees with the published data [2]. Table 3 lists the inhibitors that are derivatives of completely alkylated (peralkylated) onium ions—sulfonium (compounds 13–17), ammonium (compounds 18–21), and phosphonium (compound 22). An increase in the volume of onium ions (owing to elimination of alkyl radicals) led to a monotonic increase in their effectiveness with respect to acetylcholinesterase and butyrylcholinesterase. Note that acetylcholinesterase was more sensitive to the ammonium compounds than to the sulfonium ones, which might be due to asymmetry of the hydration shell [8, 14], whereas tetrabutylphosphonium (compound 22) was closer to its sulfonium analogue (compound 16) than to the ammonium analogue (compound 21). Regarding the type of inhibitory effect, a certain difference between acetylcholinesterase and butyrylcholinesterase was revealed: in the case of acetylcholinesterase, the compounds behaved as mixed–noncompetitive inhibitors, whereas in the case of butyrylcholinesterase, the competitive type predominated (which is not characteristic of this enzyme [1, 2]).
In comparing compounds 1.2 and 1.6 (Table 1), we performed quantum chemical calculations of the electron densities on the atoms comprising their molecules. It was found that the constancy of charges on the atoms of the acyl moiety is often indicative of equal strength of the ester bond [8]. Possibly, this trend can be extrapolated to the molecules of sulfonium (compounds 9 and 11) and ammonium (compounds 10 and 12) OPIs. Therefore, it can be postulated that sulfonium and ammonium substrates have equal or similar acylating and phosphorylating abilities; the differences in their effectiveness can be assigned to the nature and, hence, rate of sorption processes (see above). Another pattern is observed when the results of quantum mechanical calculations of the electron densities on the atoms of the cationic groups of molecules of sulfonium and ammonium substrates are analyzed [8]. Note that, in addition to the differences in charges on the onium atoms, the loss of symmetry both in the configuration of the cationic group and in the distribution of electron density on it was observed. For example, the ammonium cationic group is a sphere with a homogeneous charge distribution, whereas the sulfonium group is representative of an ellipsoid dipole [8]. This fully applies to the peralkylated reversible inhibitors: the ammonium and sulfonium derivatives (compounds 18–22) are symmetrical hydrophobic cations with a charge spread over the outer spherical surface, whereas the trialkylsulfonium ions (compounds 13–17) have an asymmetric hydration shell [8]. REFERENCES 1. Sadykov, A.S., Rozengart, E.V., Abduvakhabov, A.A., and Aslanov, Kh.A., Kholinesterazy. Aktivnyi tsentr i mekhanizm deistviya (Cholisterases: The Active Center and Mechanism of Action), Tashkent: FAN, 1976.
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8. Rozengart, E.V. and Shestakova, N.N., Neirokhimiya, 1990, vol. 9, pp. 417–426. 9. Mirzabaev, E.A., Iminov, M.T., Rozengart, E.V., et al., Dokl. Akad. Nauk Uzb. SSR, 1987, no. 1, pp. 39–41. 10. Rozengart, E.V. and Basova, N.E., Dokl. Akad. Nauk, 2004, vol. 395, no. 1, pp. 117–120 [Dokl. Biochem. Biophys. (Engl. Transl.), vol. 395, no. 1, pp. 61–65]. 11. Brestkin, A.P., Rozengart, E.V., Abduvakhabov, A.A., et al., in Struktura i funktsii khimii prirodnykh i fiziologicheski aktivnykh soedinenii (Structure and Function of the Chemistry of Natural and Physiologically Active Compounds), Tashkent: Nukus, 1990, pp. 3–57. 12. Tammelin, L.-E., Acta Chem. Scand., 1957, vol. 11, pp. 1340–1349. 13. Brestkin, A.P., Rozengart, E.V., Skvortsov, N.K., et al., Dokl. Akad. Nauk SSSR, 1984, vol. 277, no. 3, pp. 735– 738.
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