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Cocaine and butyrylcholinesterase (BChE): Determination of enzymatic parameters
Life Sciences, Vol. 58, No. 13, pp. PL 257-261, 1996 Copyright0 1996 Efsevier Science Inc. Printed in the USA. All rights mewed 0024-3205/95 $15.00 + .@I
PI1 SOO24-3205(96)00065-3
ELSEWER
PHARiU4COLOGY LETTERS Accelemted Communication
COCAINE AND BUTYBYLCHOLINESTEBASE (BChE): DETERMINATION ENZYMATIC PABAMETEBS
OF
C. Mattes’, R. Bradley, E. Slaughter, S. Browne Pharmavene, Inc. 1550 East Gude Drive Rockville, MD 20850 (Submitted
December 5, 1995; accepted December received in final form January 25, 1996)
8, 1995;
Abstract. In humans, the plasma enzyme, butyrylcholinesterase (E.C. 3.l.l.Q metabolizes cocaine to the water-soluble, pharmacologically inactive compounds, ecgonine methylester and benzoic acid. Homogenous enzyme was purified from human plasma and used to determine the enzyme kinetic parameters of K, and V, with cocaine as the substrate. The Kkl (11.9 PM) indicates that cocaine is tightly bound to the four active sites of the native tetramer. The V, (1.17 pM/min) is 50-fold greater than cocaine catalytic antibodies. Administration of purified human butyrylcholinesterase to a cocaine-intoxicated patient would be expected to shift the metabolism to the inactive metabolites and reduce the toxicity. Key War+: butyrylcholinesterase,
cocaine, hydrolysis, K,,,, specific activity
Introduction In humans, the metabolism of cocaine by the plasma enzyme butyrycholinesterase (BChE) is well established (1, 2, 3). The enzymatic products from catalysis of cocaine by BChE are benzoic acid and ecgonine methylester, both pharmacologically inactive metabolites that are rapidly excreted. The normal level of BChE varies among individuals and is dependent on age state of health, exposure to environmental toxins and genetic factors. Low levels of BChE are associated with cocaine-induced cardiac and related complications (4, 5). Furthermore, deficiency of BChE shifts the metabolism of cocaine to norcocaine, a metabolite with anesthetic and cardiotoxic properties and benzoylecgonine, a metabolite that causes vasoconstriction (4,6). If metabolism of cocaine were shifted to ecgonine methylester by the administration of exogenous purified BChE, the incidence of cocaine-induced complications may in turn be reduced. To this end, purified human BChE has been developed as a therapy for cocaine intoxication. In this report, the enzyme kinetic parameters of purified human BChE with cocaine as the substrate are presented. ’ Corresponding author: Carol E. Mattes, Pharmavene, Inc., 1550 East Gude Drive, Rockville, MD 20850. Telephone: (301) 838-2562, Fax: (301) 838-2502.
PL-258
Vol. 58, No. 13,19%
Enzyme Kinetics of Pure Butyylcholinesterase
Methods Purification of BChE : BChE (EC 3.1.1.8) was purified from human plasma Cohn Fraction IV-4 (American Red Cross) by modifications to previously described anion exchange column chromatography and procainamide-Sepharose affmity chromatography (7). Preparations from 5 kg of plasma paste yielded 100 to 150 mg of homogenous enzyme as judged by SDS-PAGE and the specific activity (7). Protein concentrations were determined by a modified Lowry protein determination. During the purification procedure, enzyme activity was determined from the rate of the hydrolysis of benzoylcholine as previously described (8).
Enzyme-Substrate incubation: A stock solution of BChE was prepared by diluting the enzyme in 67 mM phosphate buffer (pH 7.4) with 0.2 mghnL BSA. The reaction was initiated by the addition of BChE (17 ug/ml) to cocaine (2.9 to 29.4 PM) and allowed to proceed for 10 min at 37°C. The reaction was quenched by the addition of saturated NaF and putting the sample on ice. The incubation mixture was directly injected onto the HPLC without further treatment.
HPLC Analysis described (9).
of Cocaine:
Cocaine was analyzed by HPLC and UV detection as previously
Results and Discussion The Michaelis-Menton constant (Km> and the maximum velocity (V,) were determined with three different lots of purified human BChE. The kinetic parameters were determined by two graphical methods, Lineweaver-Burke and Eadie-Hofstee plots, which yielded linear plots with nearly equivalent kinetic values (Fig. 1). Since formation of benzoylecgonine was insignificant under assay conditions, calculations were based upon the hydrolysis of cocaine without correction. From the Lineweaver-Burke plot, the K,, a measure of the binding affinity for the substrate,
was 11.9 + 1.18 micromolar
and the value of V,,
was determined
as 1.17 f 0.16
micromolar/min. From the V_, the specific activity was calculated as 69.7 nanomoles/minImg. The turnover number, defined as moles of substrate/min/mol of active site, was calculated to be 24 min-’ .
The pharmacokinetics of cocaine are affected by the enzymatic kinetics of BChE. The dosage of purified enzyme required for detoxification of cocaine is directly related to the turnover of the enzyme and specific activity of the preparation. Gatley (1990) did not determine Kk( and V, for (-)-cocaine due to limitations of the spectrophotometric assay (10). Stewart, et al. (1979) estimated the kinetic parameters of BChE from the hydrolysis of cocaine in unfractionated plasma (3). The K, for homogenous BChE (11.9 uM) vs. plasma (=50 uM) indicates a tighter binding of the cocaine in the enzyme active site than reported earlier (3). Similarly, the purified enzyme has a 50- to lOO-fold higher affinity for cocaine than do the catalytic antibodies (11). Moreover, the turnover of the enzyme is SO- to 500-fold greater than either antibody, respectively. Therefore, stoichiometric dosing of the enzyme is unnecessary in contrast to the catalytic antibodies (11).
-2
-0.2
PL-259
Enzyme Kinetics of Pure Butyrylchohesterase
Vol. 58, No. 13, 1996
-0.1
0
0.1
0.2
0.3
0.4
l/[S] (l/micromol~r
Fig. 1 A. Lineweaver-Burke Plot (l/V vs. l/[S]) of cocaine hydrolysis by BChE.
kz E+S-ES-EKn
A mechanism for the catalysis of benzoylcholine by BChE was proposed by Locloidge and LaDu (1978). A Michaelis complex, ES, is formed in a rapid equilibrium that is defined by the dissociation constant. The enzyme is carbamylated with a rate constant, k2, to give a carbamyl enzyme intermediate containing a covalent bond between the active site serine and carbamic acid. The release of the carbamylate intermediate (k,) is the rate limiting step of the catalytic reaction (7). The mechanism for the catalysis of cocaine by BChE should be similar although the rate constant are different. The high binding affinity for cocaine (i.e., low Km) also indicates a rapid and stable formation of the enzyme-substrate complex (K&s) while the turnover number indicates k,
Enzyme Kinetics of Pure Butyrylcholinederase
PL-260
VoL 58, No. 13,1%
0.6 -
0.00
a02
004
0.06
0.08
0.10
0.12
V/[S I (min) Fig. 1 B. Eadie-Hoffstee Plot (V/[S] vs. V) of cocaine hydrolysis by BChE. Average of three lots shown with standard error.
The value of V,, was 3- to 4-fold greater than previously determined by Stewart, et al. (1979) while the specific activity and turn over number were uniquely determined. The higher value of V_ was expected since the enzyme is homogenous and devoid of interfering or inhibitory substances (3). Furthermore, the contribution from variant forms of BChE is insignificant since the purified enzyme has been characterized as the native BChE by dibucaine and fluoride number as well as the specific activity with benzoylcholine, butyrythiocholine and propionylthiocholine as substrates (R. Bradley, unpublished data).
BChE has a good potential for use as a therapy for cocaine intoxication and would reduce the In vitro studies in total body burden of the drug by the metabolism of cocaine in the blood human plasma have demonstrated the rate of elimination is increased with addition of purified BChE (12). Thus, a doubling of the normal enzyme level from 5 mg/L to 10 mgIL would increase the rate of elimination of cocaine from the body. The high binding affinity (low K,) indicates Additional benefits from that BChE may sequester or bind the cocaine in the blood. administration of the enzyme may also result from the metabolism of norcocaine to the inactive products, norecgonine methylester and benzoic acid (3), although the enzyme kinetic parameters for this reaction with homogenous BChE were not determined in this study. After administration of exogenous BChE, the level of norcocaine, the product of N-demethylation in the liver, may be decreased by mass action since metabolism is shifted to the formation of ecgonine methylester.
Vol. 58, No. 13, 19%
Enzyme Kinetics of Pure Butyrylcholinesteras
PL-261
Alternatively aand more likely, since norcocaine is a substrate the exogenous BChE, it would be rapidly hydrolyzed. Furthermore, BChE could compete norcocaine and cocaine from the sodium channels of the heart since the Kh(of cocaine binding to the enzyme is on the same order of the Kn of cocaine and norcocaine from the sodium channel (13). Both catalytic products ecgonine methylester and norecgonine methylester are rapidly cleared by the kidney (14). Finally, as the cocaine in the blood is metabolized by the exogenous BChE, a concentration gradient between the blood and lipophilic tissues such as the brain would remain and provide a driving force to further reduce the levels of cocaine in the central nervous system. Acknowledgments Our appreciation goes to Dr. William N. Drohan of the American Red Cross for gift of the human plasma Cohn Fraction IV-4. This work was supported, in part, by SBIR grant 2R44-DA0700702A1 from the National Institute on Drug Abuse.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
D.J. STEWART, T. INABA, T., B.K. TANG, W. KALOW. Life Sci. -20 1557-1564 (1977). T. INABA, D.J. STEWART, W. KALOW. Clin. Pharm. Ther. 23 547-552 (1978). D.J. STEWART, T. INABA, M. LUCASSEN, W. KALOW. Cg. Pharm. Ther. 25 464468 (1979). A. OM, S. ELLAHHAM, J.P.ORNATO, C. PICONE, J. THEOGARAJ, G.P. CORRETJER,G.W. VETROVEC. Am. Heart J 125 1114-1117(1993). R.S. HOFFMAN, G.C. HENRY, P.M. WAX, R.rWEISMAN, M.A.HOWLAND, L.R. GOLDFRANK. J. Pharm. Exper. Ther. 263 698-702 (1992). J. KAMBAM, B. METS, R.M. HICKMG, P. JANICKI, M.F.M. JAMES, B. FULLER, R.E. KIRSCH. J. Lab. Clin. Med. 120 323-328 (1992). 0. LOCKRIDGE and B.N. LADU?Bio. Chem. 253 361-366 (1978). W. KALOW and K. GENEST. Can. J. Biochem. Physio. 35 359-346 (1957). P. BOUIS, G. TACCARD, U.A. BOELSTERLI. J. Chrom. 526 447-459 (1990). S.J. GATLEY. Biochem. Pharm. -41 1249-1254 (1991). D.W. LANDRY, K.ZHAO, G. YANG, M. GLICKMAN, T.M. GEORGIADIS. Science 259 1899-1901 (1993). S.P. BROWNE, E.A. SLAUGHTER, E.M. RUDNIC, R.A. COUCH, A.M. MCLEAN, Pharmaceutical Research 12 S374 (1995). W. CRUMB and C.W. CLARKSON. J. Pharm. Exper. Ther. 261 910-917 (1992). J. AMBRE, T.I. RUO, J. NELSON, S. BELKNAP. J. Anal. TzcoL 12 301-306.
PI1 SOO24-3205(96)00065-3
ELSEWER
PHARiU4COLOGY LETTERS Accelemted Communication
COCAINE AND BUTYBYLCHOLINESTEBASE (BChE): DETERMINATION ENZYMATIC PABAMETEBS
OF
C. Mattes’, R. Bradley, E. Slaughter, S. Browne Pharmavene, Inc. 1550 East Gude Drive Rockville, MD 20850 (Submitted
December 5, 1995; accepted December received in final form January 25, 1996)
8, 1995;
Abstract. In humans, the plasma enzyme, butyrylcholinesterase (E.C. 3.l.l.Q metabolizes cocaine to the water-soluble, pharmacologically inactive compounds, ecgonine methylester and benzoic acid. Homogenous enzyme was purified from human plasma and used to determine the enzyme kinetic parameters of K, and V, with cocaine as the substrate. The Kkl (11.9 PM) indicates that cocaine is tightly bound to the four active sites of the native tetramer. The V, (1.17 pM/min) is 50-fold greater than cocaine catalytic antibodies. Administration of purified human butyrylcholinesterase to a cocaine-intoxicated patient would be expected to shift the metabolism to the inactive metabolites and reduce the toxicity. Key War+: butyrylcholinesterase,
cocaine, hydrolysis, K,,,, specific activity
Introduction In humans, the metabolism of cocaine by the plasma enzyme butyrycholinesterase (BChE) is well established (1, 2, 3). The enzymatic products from catalysis of cocaine by BChE are benzoic acid and ecgonine methylester, both pharmacologically inactive metabolites that are rapidly excreted. The normal level of BChE varies among individuals and is dependent on age state of health, exposure to environmental toxins and genetic factors. Low levels of BChE are associated with cocaine-induced cardiac and related complications (4, 5). Furthermore, deficiency of BChE shifts the metabolism of cocaine to norcocaine, a metabolite with anesthetic and cardiotoxic properties and benzoylecgonine, a metabolite that causes vasoconstriction (4,6). If metabolism of cocaine were shifted to ecgonine methylester by the administration of exogenous purified BChE, the incidence of cocaine-induced complications may in turn be reduced. To this end, purified human BChE has been developed as a therapy for cocaine intoxication. In this report, the enzyme kinetic parameters of purified human BChE with cocaine as the substrate are presented. ’ Corresponding author: Carol E. Mattes, Pharmavene, Inc., 1550 East Gude Drive, Rockville, MD 20850. Telephone: (301) 838-2562, Fax: (301) 838-2502.
PL-258
Vol. 58, No. 13,19%
Enzyme Kinetics of Pure Butyylcholinesterase
Methods Purification of BChE : BChE (EC 3.1.1.8) was purified from human plasma Cohn Fraction IV-4 (American Red Cross) by modifications to previously described anion exchange column chromatography and procainamide-Sepharose affmity chromatography (7). Preparations from 5 kg of plasma paste yielded 100 to 150 mg of homogenous enzyme as judged by SDS-PAGE and the specific activity (7). Protein concentrations were determined by a modified Lowry protein determination. During the purification procedure, enzyme activity was determined from the rate of the hydrolysis of benzoylcholine as previously described (8).
Enzyme-Substrate incubation: A stock solution of BChE was prepared by diluting the enzyme in 67 mM phosphate buffer (pH 7.4) with 0.2 mghnL BSA. The reaction was initiated by the addition of BChE (17 ug/ml) to cocaine (2.9 to 29.4 PM) and allowed to proceed for 10 min at 37°C. The reaction was quenched by the addition of saturated NaF and putting the sample on ice. The incubation mixture was directly injected onto the HPLC without further treatment.
HPLC Analysis described (9).
of Cocaine:
Cocaine was analyzed by HPLC and UV detection as previously
Results and Discussion The Michaelis-Menton constant (Km> and the maximum velocity (V,) were determined with three different lots of purified human BChE. The kinetic parameters were determined by two graphical methods, Lineweaver-Burke and Eadie-Hofstee plots, which yielded linear plots with nearly equivalent kinetic values (Fig. 1). Since formation of benzoylecgonine was insignificant under assay conditions, calculations were based upon the hydrolysis of cocaine without correction. From the Lineweaver-Burke plot, the K,, a measure of the binding affinity for the substrate,
was 11.9 + 1.18 micromolar
and the value of V,,
was determined
as 1.17 f 0.16
micromolar/min. From the V_, the specific activity was calculated as 69.7 nanomoles/minImg. The turnover number, defined as moles of substrate/min/mol of active site, was calculated to be 24 min-’ .
The pharmacokinetics of cocaine are affected by the enzymatic kinetics of BChE. The dosage of purified enzyme required for detoxification of cocaine is directly related to the turnover of the enzyme and specific activity of the preparation. Gatley (1990) did not determine Kk( and V, for (-)-cocaine due to limitations of the spectrophotometric assay (10). Stewart, et al. (1979) estimated the kinetic parameters of BChE from the hydrolysis of cocaine in unfractionated plasma (3). The K, for homogenous BChE (11.9 uM) vs. plasma (=50 uM) indicates a tighter binding of the cocaine in the enzyme active site than reported earlier (3). Similarly, the purified enzyme has a 50- to lOO-fold higher affinity for cocaine than do the catalytic antibodies (11). Moreover, the turnover of the enzyme is SO- to 500-fold greater than either antibody, respectively. Therefore, stoichiometric dosing of the enzyme is unnecessary in contrast to the catalytic antibodies (11).
-2
-0.2
PL-259
Enzyme Kinetics of Pure Butyrylchohesterase
Vol. 58, No. 13, 1996
-0.1
0
0.1
0.2
0.3
0.4
l/[S] (l/micromol~r
Fig. 1 A. Lineweaver-Burke Plot (l/V vs. l/[S]) of cocaine hydrolysis by BChE.
kz E+S-ES-EKn
A mechanism for the catalysis of benzoylcholine by BChE was proposed by Locloidge and LaDu (1978). A Michaelis complex, ES, is formed in a rapid equilibrium that is defined by the dissociation constant. The enzyme is carbamylated with a rate constant, k2, to give a carbamyl enzyme intermediate containing a covalent bond between the active site serine and carbamic acid. The release of the carbamylate intermediate (k,) is the rate limiting step of the catalytic reaction (7). The mechanism for the catalysis of cocaine by BChE should be similar although the rate constant are different. The high binding affinity for cocaine (i.e., low Km) also indicates a rapid and stable formation of the enzyme-substrate complex (K&s) while the turnover number indicates k,
Enzyme Kinetics of Pure Butyrylcholinederase
PL-260
VoL 58, No. 13,1%
0.6 -
0.00
a02
004
0.06
0.08
0.10
0.12
V/[S I (min) Fig. 1 B. Eadie-Hoffstee Plot (V/[S] vs. V) of cocaine hydrolysis by BChE. Average of three lots shown with standard error.
The value of V,, was 3- to 4-fold greater than previously determined by Stewart, et al. (1979) while the specific activity and turn over number were uniquely determined. The higher value of V_ was expected since the enzyme is homogenous and devoid of interfering or inhibitory substances (3). Furthermore, the contribution from variant forms of BChE is insignificant since the purified enzyme has been characterized as the native BChE by dibucaine and fluoride number as well as the specific activity with benzoylcholine, butyrythiocholine and propionylthiocholine as substrates (R. Bradley, unpublished data).
BChE has a good potential for use as a therapy for cocaine intoxication and would reduce the In vitro studies in total body burden of the drug by the metabolism of cocaine in the blood human plasma have demonstrated the rate of elimination is increased with addition of purified BChE (12). Thus, a doubling of the normal enzyme level from 5 mg/L to 10 mgIL would increase the rate of elimination of cocaine from the body. The high binding affinity (low K,) indicates Additional benefits from that BChE may sequester or bind the cocaine in the blood. administration of the enzyme may also result from the metabolism of norcocaine to the inactive products, norecgonine methylester and benzoic acid (3), although the enzyme kinetic parameters for this reaction with homogenous BChE were not determined in this study. After administration of exogenous BChE, the level of norcocaine, the product of N-demethylation in the liver, may be decreased by mass action since metabolism is shifted to the formation of ecgonine methylester.
Vol. 58, No. 13, 19%
Enzyme Kinetics of Pure Butyrylcholinesteras
PL-261
Alternatively aand more likely, since norcocaine is a substrate the exogenous BChE, it would be rapidly hydrolyzed. Furthermore, BChE could compete norcocaine and cocaine from the sodium channels of the heart since the Kh(of cocaine binding to the enzyme is on the same order of the Kn of cocaine and norcocaine from the sodium channel (13). Both catalytic products ecgonine methylester and norecgonine methylester are rapidly cleared by the kidney (14). Finally, as the cocaine in the blood is metabolized by the exogenous BChE, a concentration gradient between the blood and lipophilic tissues such as the brain would remain and provide a driving force to further reduce the levels of cocaine in the central nervous system. Acknowledgments Our appreciation goes to Dr. William N. Drohan of the American Red Cross for gift of the human plasma Cohn Fraction IV-4. This work was supported, in part, by SBIR grant 2R44-DA0700702A1 from the National Institute on Drug Abuse.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
D.J. STEWART, T. INABA, T., B.K. TANG, W. KALOW. Life Sci. -20 1557-1564 (1977). T. INABA, D.J. STEWART, W. KALOW. Clin. Pharm. Ther. 23 547-552 (1978). D.J. STEWART, T. INABA, M. LUCASSEN, W. KALOW. Cg. Pharm. Ther. 25 464468 (1979). A. OM, S. ELLAHHAM, J.P.ORNATO, C. PICONE, J. THEOGARAJ, G.P. CORRETJER,G.W. VETROVEC. Am. Heart J 125 1114-1117(1993). R.S. HOFFMAN, G.C. HENRY, P.M. WAX, R.rWEISMAN, M.A.HOWLAND, L.R. GOLDFRANK. J. Pharm. Exper. Ther. 263 698-702 (1992). J. KAMBAM, B. METS, R.M. HICKMG, P. JANICKI, M.F.M. JAMES, B. FULLER, R.E. KIRSCH. J. Lab. Clin. Med. 120 323-328 (1992). 0. LOCKRIDGE and B.N. LADU?Bio. Chem. 253 361-366 (1978). W. KALOW and K. GENEST. Can. J. Biochem. Physio. 35 359-346 (1957). P. BOUIS, G. TACCARD, U.A. BOELSTERLI. J. Chrom. 526 447-459 (1990). S.J. GATLEY. Biochem. Pharm. -41 1249-1254 (1991). D.W. LANDRY, K.ZHAO, G. YANG, M. GLICKMAN, T.M. GEORGIADIS. Science 259 1899-1901 (1993). S.P. BROWNE, E.A. SLAUGHTER, E.M. RUDNIC, R.A. COUCH, A.M. MCLEAN, Pharmaceutical Research 12 S374 (1995). W. CRUMB and C.W. CLARKSON. J. Pharm. Exper. Ther. 261 910-917 (1992). J. AMBRE, T.I. RUO, J. NELSON, S. BELKNAP. J. Anal. TzcoL 12 301-306.