Inhibition of human erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7) and serum butyrylcholinesterase (BChE; EC 3.1.1.8) by ethopropazine, 10-(2-diethylaminopropyl)phenothiazine hydrochloride, was measured with acetylthiocholine (ATCh) as substrate. Dissociation constants for the enzyme-inhibitor complexes were calculated from the effect of ATCh concentration on the apparent dissociation constants by applying non-linear regression to fit the model to experimental data. Inhibition of AChE revealed a competitive inhibition for two binding sites (Ka = 161 and Ki = 393 &mu;mol dm-3), inhibition of the atypical BChE was non-competitive (Ki = 7.5 &mu;mol dm-3) while that of the usual BChE was competitive (K(I) = 0.16 &mu;mol dm-3). At the ethopropazine concentration of 20 &mu;mol dm-3 and the acetyl-thiocholine concentration of 1.0 mmol dm-3 (conditions used for differentiation between AChE and BChE activities), the erythrocyte AChE was 8% inhibited and the BChE phenotypes UU, UA, FF/FS, AF, AJ/AK and AA/AS between 98% and 74%

Anita Štuglin

Elsa Reiner

Goran Šinko

Vera Simeon-Rudolf

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ISSN-0011-1643CCA-2723 Original Scientific PaperInhibition of Human Blood Acetylcholinesteraseand Butyrylcholinesterase by EthopropazineVera Simeon-Rudolf,* Goran [inko, Anita [tuglin, and Elsa ReinerInstitute for Medical Research and Occupational Health,Ksaverska cesta 2, 10000 Zagreb, CroatiaReceived August 24, 2000; revised November 2, 2000; accepted November 3, 2000Inhibition of human erythrocyte acetylcholinesterase (AChE; EC3.1.1.7) and serum butyrylcholinesterase (BChE; EC 3.1.1.8) by etho-propazine, 10-(2-diethylaminopropyl)phenothiazine hydrochloride, wasmeasured with acetylthiocholine (ATCh) as substrate. Dissociationconstants for the enzyme-inhibitor complexes were calculated fromthe effect of ATCh concentration on the apparent dissociation con-stants by applying non-linear regression to fit the model to experi-mental data. Inhibition of AChE revealed a competitive inhibitionfor two binding sites (Ka = 161 and Ki = 393 mol dm–3), inhibitionof the atypical BChE was non-competitive (Ki = 7.5 mol dm–3) whilethat of the usual BChE was competitive (K(I) = 0.16 mol dm–3).At the ethopropazine concentration of 20 mol dm–3 and the acetyl-thiocholine concentration of 1.0 mmol dm–3 (conditions used for dif-ferentiation between AChE and BChE activities), the erythrocyteAChE was 8% inhibited and the BChE phenotypes UU, UA, FF/FS,AF, AJ/AK and AA/AS between 98% and 74%.Key words: acetylcholinesterase, butyrylcholinesterase, ethopropa-zine, selective inhibition, inhibition mechanism, enzyme-inhibitorand enzyme-substrate dissociation constants.INTRODUCTIONEthopropazine is one of the phenothiazine derivatives that are potent in-hibitors of butyrylcholinesterase (BChE, EC 3.1.1.8), but poor inhibitors ofacetylcholinesterase (AChE, EC 3.1.1.7). As the most potent and selective* Author to whom correspondence should be addressed. (E-mail: vesimeon@imi.hr)CROATICA CHEMICA ACTA CCACAA 74 (1) 173¿182 (2001)inhibitor among phenothiazine derivatives, ethopropazine is used to inhibitselectively BChE in samples where both BChE and AChE are present.1–4Ethopropazine has a quaternary nitrogen in the structure (Figure 1), andone may expect that it inhibits the usual human serum BChE to a higherdegree than the atypical enzyme or its heterozygotes. We confirmed this as-sumption in a preliminary study.5This paper deals with the inhibition of human serum BChE variants andof human erythrocyte AChE by racemic ethopropazine. The selectivity of etho-propazine for different human BChE phenotypes and for human erythrocyteAChE was determined under the experimental conditions used for assess-ment of AChE and BChE activities in human blood. To get more informationon the inhibition mechanism of the two enzymes, the dissociation constantsof the enzyme-inhibitor complexes were determined from the effect of thesubstrate upon the degree of inhibition.EXPERIMENTALEnzyme SourceThe source of AChE was human erythrocytes washed with buffer to remove theplasma. Sera were taken from individuals whose BChE phenotypes had previouslybeen determined by measuring the activity with propionylthiocholine as substrateand the inhibition with benzoylcholine as substrate using dibucain, sodium fluorideand Ro 02-0683 as differentiating inhibitors.6–8 The phenotypes were homozygotes orheterozygotes of the usual (U), atypical (A), fluoride-resistant (F), Kalow (K), J, or si-lent (S) BChE variants.174 V. SIMEON-RUDOLF ET AL.NSCH2HC CH3HNH C C HCl22 55Figure 1. Chemical structure of ethopropazine.ReagentsEthopropazine (Sigma, St. Louis, USA) was a gift from Professor P. Eyer. Acetyl-thiocholine iodide (ATCh) was from Sigma St. Louis, USA.Activity MeasurementActivity of the enzymes was measured with ATCh in 0.1 mol dm–3 phosphate buffer,pH = 7.4 at 37 C with the thiol reagent DTNB.9 In the final reaction mixture, erythro-cytes and serum were diluted 600 and 150 times, respectively. Activities measuredat ATCh concentrations of 0.5 mmol dm–3 or higher were corrected for spontaneoussubstrate hydrolysis. More details of the procedure were described earlier.10,11Enzyme InhibitionThe selectivity of ethopropazine for AChE and BChE was tested with ethopro-pazine concentration of 20 mol dm–3 and ATCh concentration of 1.0 mmol dm–3. Tostudy the competition between ethopropazine and the substrate, the activity of AChEand BChE was measured in the absence and presence of ethopropazine with ATChconcentrations ranging between 0.1 and 10 mmol dm–3. From experimental data thusobtained, enzyme-inhibitor and enzyme-substrate dissociation constants were calcu-lated from the equations, based on the assumption that the enzyme has a peripheral(allosteric) site where substrates or inhibitors can bind.13If both, substrate and inhibitor, bind to both sites on the enzyme (catalytic andperipheral), the equation which relates substrate and inhibitor concentrations to en-zyme activities is:13Ki K s s KK s K K iapps sss a i  nn n01 11 1( / ) ( / )( / ) ( / / / (K K K K K Ka i i s a ss)) / / ( ) 1(1)where 0 and  are the enzyme activities at a given substrate concentration (s) in theabsence and presence of the inhibitor (concentration: i). In this equation, the appar-ent dissociation constant Kapp is a function of both inhibitor and substrate concentra-tions. The Ks, Kss, Ka and Ki are dissociation constants of enzyme-substrate and en-zyme-inhibitor complexes at the catalytic and peripheral sites, respectively.If the inhibitor binds only to the catalytic site or only to the peripheral site, andthe substrate binds to both sites, the effect of substrate concentration upon Kapp isexpressed by the following equation:13KiKKKsapp IIS nn n0( )( )( )(2)K(I) is either the enzyme-inhibitor dissociation constant at the catalytic site (Ka)or the dissociation constant at the peripheral site (Ki), and K(S) is the enzyme-sub-strate dissociation constant at the respective sites (Ks or Kss). It follows from Eq. (2)that Kapp is only a function of the substrate concentration.CHOLINESTERASE INHIBITION BY ETHOPROPAZINE 175Finally, if Kapp does not change with the change of substrate concentration, anon-competitive inhibition occurs, and consequentlyKiKapp inn n0(3)The enzyme-inhibitor and enzyme-substrate dissociation constants were calcu-lated from the above equations by the MacCurvefit Programme applying the Quasi-Newton minimisation algorithm.RESULTSSelectivity of Ethopropazine for Acetylcholinesteraseand ButyrylcholinesteraseAbsorption of cholinesterase inhibitors is commonly assessed by measur-ing the decrease of AChE and/or BChE activities in human blood. If activi-ties are measured in whole blood, i.e. without separating the erythrocytesfrom plasma, one should apply either a specific substrate or a specific inhibi-tor of AChE or BChE in order to assess the activity of only one enzyme. Etho-propazine has been suggested as a specific BChE inhibitor, which at the con-centration of 20 mol dm–3 fully inhibits the BChE activity without inhibitingAChE, and procedures have been worked out for the differentiation of thetwo enzymes in whole blood.2,3In this paper we have tested ethopropazine as an inhibitor of BChE va-riants and of erythrocyte AChE. Inhibition was measured with the ethopro-pazine concentration of 20 mol dm–3 using ATCh (1.0 mmol dm–3) as sub-strate (Table I).The mean activities (mol min–1 mL–1) of the studied BChE variantsranged from 2.86 for the usual BChE (UU) to 0.41 for the atypical enzyme(AA/AS). All six variants were inhibited by the ethopropazine concentrationof 20 mol dm–3. The degree of inhibition decreased in almost the same or-der as the rates of ATCh hydrolysis: the UU BChE, which is most active,was 98% inhibited while the AA/AS BChE was only 74% inhibited. Underthe same experimental conditions, ethopropazine (20 mol dm–3) inhibited8% of erythrocyte AChE, which is consistent with 5% inhibition reported byWorek et al.3Consequently, ethopropazine is a very potent inhibitor of BChE. How-ever, when ethopropazine is applied as inhibitor of BChE in order to mea-sure the AChE activity in human whole blood, AChE will also be slightlyinhibited. On the other hand, when blood contains a homozygous or hetero-zygous A variant of BChE, inhibition of the serum BChE by ethopropazine176 V. SIMEON-RUDOLF ET AL.(20 mol dm–3) will be less than 98%. Both of these facts should be takeninto account when validating procedures for the AChE activity measure-ments in whole blood, based on BChE inhibition by ethopropazine.12Reversible Binding of Ethopropazine to Acetylcholinesteraseand ButyrylcholinesteraseTo determine the dissociation constants of the ethopropazine complexeswith AChE or BChE, inhibition was measured with a broad range of etho-propazine concentrations in order to reach a degree of inhibition between 20to 80%. Experimental data were interpreted according to the models describedby Eqs. (1–3).The apparent dissociation constants Kapp for inhibition of AChE and BChEas a function of the ATCh concentration (Hunter-Downs plots) are shown inFigure 2.The Hunter-Downs plot for the inhibition of erythrocyte AChE displayeda curve. Applying Eq. (1), a theoretical curve and the best fitting dissociationconstants Ks, Kss, Ka and Ki were calculated (Table II). The curve fitting wasdone for the ethopropazine concentration of 200 mol dm–3 because that con-centration was used in the experiments. The theoretical curves calculatedfor ethopropazine concentrations of 200 and 400 mol dm–3 fell within onestandard deviation of the experimental points. The calculated Ks and Kssvalues (Table II) are in good agreement with the Km and Kss constants weCHOLINESTERASE INHIBITION BY ETHOPROPAZINE 177TABLE IActivities of AChE and BChE, and inhibition of cholinesterases by ethopropazine(20 mol dm–3), measured with ATCh (1.0 mmol dm–3)Enzyme NActivitymolmin mLa–1–1Inhibition%aErythrocyte AChE 6 4.66  1.08 08  4Serum BChE phenotypeUU 4 2.86  0.57 98  2UA 4 1.92  0.33 94  1FF/FS 5 0.66  0.11 92  1AF 3 0.85  0.26 87  1AJ/AK 6 0.51  0.14 85  3AA/AS 5 0.41  0.04 74  4a Mean  SD. Mean values were calculated from measurements done on erythrocytes or serafrom N individuals.178 V. SIMEON-RUDOLF ET AL.Figure 2. Inhibition of erythrocyte acetylcholinesterase (AChE) and serum butyryl-cholinesterase (BChE) variants (UU and AA/AS) by ethopropazine, measured withacetylthiocholine (ATCh) as substrate. Kapp is defined by Eqs. (1), (2) or (3). Eachpoint is the mean value of 4–7 experiments. The lines were calculated as describedunder Experimental.calculated from the data for erythrocyte AChE and ATCh in the absence ofethopropazine (0.1 and 5.7 mmol dm–3, respectively).The Hunter-Downs plot for the usual BChE displayed competitive inhi-bition and Eq. (2) was therefore applied (Table II). However, the Kapp valuesmeasured at ATCh concentrations of 0.10 and 0.25 mmol dm–3 were almostthe same, 0.19 and 0.20 mol dm–3 respectively, indicating a non-competi-tive component of inhibition at low substrate concentrations. As the K(I) con-stant derived from Eq. (2) (Table II) was not significantly different from theabove two Kapp values, we find it justified to attribute only one enzyme-in-hibitor dissociation constant to the inhibition of the usual BChE by etho-propazine. The value of the K(S) constant derived from Eq. (2) (Table II) wasbetween two enzyme-substrate dissociation constants calculated in the ab-sence of ethopropazine either from data in this paper (K1 = 0.04 mmol dm–3and K2 = 1.2 mmol dm–3) or in a previous study (K1 = 0.04 mmol dm–3 andK2 = 1.8 mmol dm–3).10 These enzyme-substrate dissociation constants werecalculated by applying an equation which assumes two binding sites for thesubstrate on the enzyme.14Inhibition of the atypical BChE was non-competitive over the totalATCh concentration range studied. This follows from Figure 2 and also fromthe analysis of Kapp values at ATCh concentrations of 0.10 and 10 mmol dm–3,which were not significantly different (t = 1.99, at the 0.094 significance le-vel). The non-competitive inhibition constant Ki was therefore calculated asthe mean of all the measured Kapp values according to Eq. (3) (Table II). Ac-cording to the model underlying Eqs. (1–3), the non-competitive feature ofinhibition points to the involvement of the peripheral site in the inhibitionmechanism.CHOLINESTERASE INHIBITION BY ETHOPROPAZINE 179TABLE IIDissociation constants for enzyme-inhibitor and enzyme-substrate complexescalculated by Eqs. (1), (2) or (3) from Kapp values shown in Figure 2EnzymeATChmmol dm–3Ethopropazinemol dm–3mInhibitor constantsmol dm–3mSubstrate constantsmmol dm–3ErythrocyteAChE 0.1–10 200Ka = 161Ki = 393Ks = 0.10Kss = 7.3UU phenotypeBChE0.1–10 0.25–2 K(I) = 0.16  0.03 K(S) = 0.69  0.14AA phenotypeBChE0.1–10 2–10 Ki = 7.5  1.4 –DISCUSSION AND CONCLUSIONDifferentiation between AChE and BChE activities is routinely done withethopropazine (20 mol dm–3) as a potent BChE inhibitor, and the activitiesare measured with ATCh (1 mmol dm–3) as substrate. It follows from theevaluated enzyme-inhibitor dissociation constants given in Table II that underthese conditions the erythrocyte AChE and the usual BChE variant wouldbe inhibited 6% and 98%, respectively. The atypical BChE variant would beinhibited 73% at any ATCh concentration between 0.1 and 10 mmol dm–3.The measured percents of inhibition given in Table I agree well with thesevalues, calculated from the enzyme-inhibitor dissociation constants. This inturn means that the equations applied to the calculation of the dissociationconstants are valid over a broad range of ethopropazine concentrations, in-cluding the concentration of 20 mol dm–3 which was not used in the experi-ments wherefrom the respective dissociation constants were calculated (Ta-ble II).The competition between ethopropazine and acetylthiocholine clearly showsa difference between AChE and BChE. AChE displays two binding sites, andATCh competes with ethopropazine at both sites. The atypical BChE displaysa non-competitive inhibition, which indicates a peripheral binding site forethopropazine. The usual BChE reveals primarily a competitive inhibition,which however has a non-competitive component at low substrate concen-trations. Attribution of only one enzyme-inhibitor dissociation constant mighttherefore be an oversimplification of the inhibition mechanism.The affinity of ethopropazine for the usual BChE is three-orders of mag-nitude higher than for the AChE. Similar differences between the inhibitionof AChE and BChE were found for enzymes from other sources. The speci-ficity of ethopropazine in interaction with cholinesterases was demonstratedthrough molecular modelling and confirmed by site specific mutagenesis. Theselectivity was related to the presence of a bulky aromatic residue, tyrosineor phenylalanine, in AChE, in place of an alanine found at the correspond-ing position in BChE. For this reason, the binding site is more spacious inBChE and is capable of admitting the bulky ethopropazine.4,15,16Positively charged compounds are better inhibitors of the usual BChEvariant than of the atypical variant,6,17 and this was also confirmed for etho-propazine (Tables I and II) which has a quaternary nitrogen in the molecule(Figure 1). The affinity of ethopropazine for the usual phenotype was about50-fold higher than for the atypical phenotype, which results from the one-point mutation in the atypical enzyme (Asp 70 Gly) as compared to theusual enzyme. The AJ/AK sera were inhibited less than the usual enzymedue to the presence of the atypical variant in heterozygotes; mutations on J180 V. SIMEON-RUDOLF ET AL.and K variants are outside the active site and they are not critical for thebinding of substrates or inhibitors.18,19Acknowledgement. – This work was supported by the Ministry of Science andTechnology of the Republic of Croatia (Grant No. 00220104).REFERENCES1. K.-B. Augustinsson, H. Eriksson, and Y. Faijersson, Clin. Chim. Acta 89 (1978)239–252.2. W. J. A. Meuling, M. J. M. Jongen, and J. J. van Hemmen, Am. J. Ind. Med. 22(1992) 231–241.3. F. Worek, U. Mast, D. Kiderlen, C. Diepold, and P. Eyer, Clin. Chim. Acta 288(1999) 73–90.4. Z. Radi}, N. A. Pickering, D. C. Vellom, S. Camp, and P. Taylor, Biochemistry 32(1993) 12074–12084.5. V. Simeon-Rudolf, G. [inko, and E. Reiner, in: I. Ferjan, M. Kr`an, L. Stanovnik,and M. ^arman-Kr`an (Eds.), Life Sciences Conference 1999. Book of Abstractsand Programme, Slovenian Pharmacological Society, Ljubljana, 1999, p. 87.6. M. Whittaker, Cholinesterase, in: L. Beckmann (Ed.), Monographs in Human Ge-netics, Vol. 11, Karger A. G., Basel, 1986, pp. 45–64.7. V. Simeon-Rudolf, Z. Kovarik, M. [krinjari}-[poljar, and R. T. Evans, Chem. Biol.Interact. 119–120 (1999) 159–164.8. V. Simeon, A. Bunti}, B. [urina, and Z. Flegar-Me{tri}, Acta Pharm. Jugosl. 37(1987) 107–114.9. G. L. Ellman, K. D. Courtney, V. Andres, and R. M. Featherstone, Biochem. Phar-macol. 7 (1961) 88–95.10. V. Simeon-Rudolf and B. Jur{i}, Period. Biol. 98 (1996) 331–335.11. E. Reiner, M. [krinjari}-[poljar, S. Dunaj, V. Simeon-Rudolf, I. Primo`i~, and S.Tomi}, Chem. Biol. Interact. 119–120 (1999) 173–181.12. E. Reiner, G. [inko, M. [krinjari}-[poljar, and V. Simeon-Rudolf, Arh. Hig. RadaToksikol. 51 (2000) 13–18.13. N. W. Aldridge and E. Reiner, Enzyme Inhibitors as Substrates. Interaction of Es-terases with Esters of Organophosphorus and Carbamic Acids, North HollandPubl. Co., Amsterdam, 1972, pp. XVI + 328.14. G. Cauet, A. Friboulet, and D. Thomas, Biochem. Biophys. Acta 912 (1987) 338–342.15. A. Saxena, A. M. G. Redman, X. Jiang, O. Lockridge, and B. P. Doctor, Biochemis-try 36 (1997) 14642–14651.16. A. Saxena, A. M. G. Redman, X. Jiang, O. Lockridge, and B. P. Doctor, Chem. Biol.Interact. 119–120 (1999) 61–69.17. E. Reiner, V. Simeon-Rudolf, and M. [krinjari}-[poljar, Toxicol. Lett. 82/83 (1995)447–452.18. O. Lockridge, Pharmac. Ther. 47 (1990)1086–1103.19. Z. Kovarik, Period. Biol. 101 (1999) 7–15.CHOLINESTERASE INHIBITION BY ETHOPROPAZINE 181SA@ETAKInhibicija acetilkolinesteraze i butirilkolinesteraze iz ljudske krvietopropazinomVera Simeon-Rudolf, Goran [inko, Anita [tuglin i Elsa ReinerInhibicija ljudske eritrocitne acetilkolinesteraze (AChE, EC 3.1.1.7) i serumskebutirilkolinesteraze (BChE, EC 3.1.1.8) etopropazinom, 10-(2-dietilaminopropil)feno-tiazin-hidrokloridom, mjerena je pri raznim koncentracijama supstrata acetiltioko-lina. Disocijacijske konstante kompleksa enzim-inhibitor izra~unane su iz utjecajasupstrata na prividne disocijacijske konstante nelinearnom regresijom, prema mo-delima definiranim teorijskim jednad`bama. Inhibicija AChE bila je kompetitivna ucijelom podru~ju koncentracije supstrata i uo~ena su dva vezna mjesta (Ka = 161 iKi = 393 mol dm–3). Inhibicija atipi~ne BChE bila je nekompetitivna (Ki = 7,5 moldm–3), a obi~ne BChE kompetitivna (K(I) = 0,16 mol dm–3). Pri koncentracijama eto-propazina od 20 mol dm–3 i acetiltiokolina od 1,0 mmol dm–3 (uvjeti kori{teni zarazlikovanje aktivnosti BChE i AChE) eritrocitna AChE bila je inhibirana 8%, a BChE(fenotipovi UU, UA, FF/FS, AF, AJ/AK i AA/AS) izme|u 98% i 74%.182 V. SIMEON-RUDOLF ET AL.

Inhibition of Human Blood Acetylcholinesterase and Butyrylcholinesterase by Ethopropazine

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