- Email: [email protected]
(23) Kinetics of hydrolysis of acetylthiocholine and acetylcholine by cholinesterases
Extended Abstracts / Chemico-Biological Interactions 157–158 (2005) 353–434 1-(4-hydroxyiminomethylpyridinium)-3-(carbamoylpyridinium)propane dibromide, Tetrahedron Lett. 44 (2003) 3123–3125. [4] K. Kuca, J. Cabal, J. Patocka, J. Kassa, Synthesis of bisquaternary symmetric – ␣,␦-bis(2-hydroxyiminomethylpyridinium)alkane dibromides and their reactivation of cyclosarin-inhibited acetylcholinesterase, Lett. Org. Chem. 1 (2004) 84–86. [5] J. Bajgar, L. Mate, Inhibition of multiple molecular forms of human brain acetylcholinesterase by different types of inhibitors, Sb. Ved. Pr. Lek. Fak. Karlovy Univerzity Hradci Kralove 32 (1989) 425–435.
permeabilities. The defect course of enzymatic hydrolysis of acetylcholine is considered to be one of the possible reasons of Alzheimer disease. 2. Theoretical part Hydrolysis of ATCH (ACH) by cholinesterases is described by the reaction course k1
doi:10.1016/j.cbi.2005.10.067
387
k2
E + S ↔ ES→E + P1 + P2 k−1
(1)
Department of Physical Chemistry, Faculty of Chemical Technology, University of Pardubice, Czech Republic
where E is enzyme (BuCHE or ACHE), S is substrate (ATCH or ACH), ES is complex substate – enzyme which falls into products of hydrolysis P1 and P2 (thiocholine (TCH) or choline (CH) and acetic acid (HA)). From general reaction scheme, Michaelis–Menten’s equation describing the decrease of S or production of P versus time is derived.
Abstract
3. Experimental methods
Kinetics of hydrolysis of acetylthiocholine (ATCH) and acetylcholine (ACH) by butyrylcholinesterase (BCHE) and acetylcholinesterase (ACHE) are studied. ATCH is used for testing of enzymatic hydrolysis of ACH in vitro, because mechanism of ATCH hydrolysis is qualitatively similar to ACH and its reaction course can be quantitatively on-line measured by two independent methods: spectrophotometrical (determination of thiocholine – product of ATCH hydrolysis – using Ellman’s method) and electrochemical (determination of acetic acid – product of ATCH hydrolysis – by pH-stat method). All tested hydrolyses correspond to the Michaelis–Menten’s equation with the second irreversible step up to the total exhaustion of the substrate. The correlations were made by means of differential and integral kinetic equations describing Michaelis–Menten model. The optimal values of Michaelis constant (KM ), maximum velocity (Vm ), kinetic constants of single reaction steps and absolute concentration of the used enzyme were calculated for each experiment.
3.1. Ellman’s spectrophotometrical method
(23) Kinetics of hydrolysis of acetylthiocholine and acetylcholine by cholinesterases A. Komersov´a ∗ , K. Komers, P. Zdraˇzilov´a
1. Introduction Acetylcholine as a neuromediator of the cholinergic nerve transfer plays a key role in the physiological function of the nervous system because the binding of ACH to the postsynaptic membrane markedly changes its ionic ∗
Corresponding author. E-mail address: [email protected] (A. Komersov´a).
Ellman’s spectrophotometrical method is based on the fact that thiocholine (one of the products of enzymatic hydrolysis of ATCH) reacts immediately, quntitatively and irreversibly with 5,5 -dithiobis-2-nitrobenzoic acic (Ellman’s reagent, DTNB) forming yellow product – 5-mercapto-2-nitrobenzoic acid and its dissociated forms at pH 8. The dependences of absorbance (A) versus time (t) were measured at pH 8 (phosphate buffer) and temperature 25 ◦ C at constant wavelenght 412 nm using UV-VIS spectrograph (8453 Agilent, HewlettPackard, USA). These dependences were transformed to the dependences of TCH concentration versus t and compared with the kinetic model (Michaelis–Menten’s equation). 3.2. pH-stat method Acetic acid (product of enzymatic hydrolysis of ATCH or ACH) decreases pH value of the reaction mixture. pH-stat (736 GP Titrino, Metrohm, Switzerland) continuous monitors pH value of the reaction mixture and at its fall automatic buret doses volumetric solution of KOH to the reaction mixture and pH value is adjusted to 8. The dependence of volume of KOH solution versus time is measured and this dependence is transformed to the dependence concentration of acetic acid [HA] versus t. From concentration of [HA] in reaction mixture versus time the kinetic model of hydrolysis ATCH can be evaluated.
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Extended Abstracts / Chemico-Biological Interactions 157–158 (2005) 353–434
Fig. 1. pH-stat method.
4. Conclusions • It was found to be that hydrolyses of ATCH + BuCHE, ATCH + ACHE and ACH + BuCHE course from start to the exhaustion of substrate by Michaelis–Menten’s equation (Briggs–Haldane) for given conditions (pH, temperature) with irreversible decay of ES complex. • Average values of KM and Vm , rate constants ki and estimations of absolute concentration of given enzyme in reaction mixture [E]0 using non-linear regression model of GraphPad PRISM and solving of the system of differencial kinetic equations by GEPASI Program were found for single combinations of S and E. Agreement of proposed models and experiment is shown at Fig. 1. Demonstration of the agreement of theoretical kinetic model (1) and experimental data. Acknowledgements This work was financially supported by Czech Ministerium of Education, Helth and Sport—Research project CZ 340003/22/3340. doi:10.1016/j.cbi.2005.10.068
(24) Acetylcholinesterase mutants: oxime-assisted catalytic scavengers of organophosphonates Zrinka Kovarik a,b,∗ , Zoran Radi´c a, Vera Simeon-Rudolf b, Elsa Reiner b, Palmer Taylor a a
Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093-0636, USA b Institute for Medical Research and Occupational Health, Ksaverska cesta 2, P.O. Box 291, HR-10001 Zagreb, Croatia 1. Introduction Reaction of organophosphonates with acetylcholinesterase (AChE; EC 3.1.1.7) is characterized by the formation of serine-conjugated adducts that are only slowly reversible. Since oximes are the only known antidote to nerve agent poisoning that restore the activity of AChE, mutagenesis of AChE should enable one to develop more effective scavenging agents in which AChE mutants in combination with an oxime would complete a catalytic cycle of hydrolysis of the organophosphate by rapid inhibition followed by rapid nucleophile-mediated hydrolysis of the phosphonyl enzyme conjugate. We enlarged the active site gorge of mouse AChE with mutations Y337A, F295L and F297I. Based on rapid inhibition of mutants with SP -cycloheptyl methylphosphonyl thiocholine (SP -CHMPTCh) [1,2] and enhance∗
Corresponding author. Tel.: +385 1 46 73 188; fax: +385 1 46 73 303. E-mail address: [email protected] (Z. Kovarik).
permeabilities. The defect course of enzymatic hydrolysis of acetylcholine is considered to be one of the possible reasons of Alzheimer disease. 2. Theoretical part Hydrolysis of ATCH (ACH) by cholinesterases is described by the reaction course k1
doi:10.1016/j.cbi.2005.10.067
387
k2
E + S ↔ ES→E + P1 + P2 k−1
(1)
Department of Physical Chemistry, Faculty of Chemical Technology, University of Pardubice, Czech Republic
where E is enzyme (BuCHE or ACHE), S is substrate (ATCH or ACH), ES is complex substate – enzyme which falls into products of hydrolysis P1 and P2 (thiocholine (TCH) or choline (CH) and acetic acid (HA)). From general reaction scheme, Michaelis–Menten’s equation describing the decrease of S or production of P versus time is derived.
Abstract
3. Experimental methods
Kinetics of hydrolysis of acetylthiocholine (ATCH) and acetylcholine (ACH) by butyrylcholinesterase (BCHE) and acetylcholinesterase (ACHE) are studied. ATCH is used for testing of enzymatic hydrolysis of ACH in vitro, because mechanism of ATCH hydrolysis is qualitatively similar to ACH and its reaction course can be quantitatively on-line measured by two independent methods: spectrophotometrical (determination of thiocholine – product of ATCH hydrolysis – using Ellman’s method) and electrochemical (determination of acetic acid – product of ATCH hydrolysis – by pH-stat method). All tested hydrolyses correspond to the Michaelis–Menten’s equation with the second irreversible step up to the total exhaustion of the substrate. The correlations were made by means of differential and integral kinetic equations describing Michaelis–Menten model. The optimal values of Michaelis constant (KM ), maximum velocity (Vm ), kinetic constants of single reaction steps and absolute concentration of the used enzyme were calculated for each experiment.
3.1. Ellman’s spectrophotometrical method
(23) Kinetics of hydrolysis of acetylthiocholine and acetylcholine by cholinesterases A. Komersov´a ∗ , K. Komers, P. Zdraˇzilov´a
1. Introduction Acetylcholine as a neuromediator of the cholinergic nerve transfer plays a key role in the physiological function of the nervous system because the binding of ACH to the postsynaptic membrane markedly changes its ionic ∗
Corresponding author. E-mail address: [email protected] (A. Komersov´a).
Ellman’s spectrophotometrical method is based on the fact that thiocholine (one of the products of enzymatic hydrolysis of ATCH) reacts immediately, quntitatively and irreversibly with 5,5 -dithiobis-2-nitrobenzoic acic (Ellman’s reagent, DTNB) forming yellow product – 5-mercapto-2-nitrobenzoic acid and its dissociated forms at pH 8. The dependences of absorbance (A) versus time (t) were measured at pH 8 (phosphate buffer) and temperature 25 ◦ C at constant wavelenght 412 nm using UV-VIS spectrograph (8453 Agilent, HewlettPackard, USA). These dependences were transformed to the dependences of TCH concentration versus t and compared with the kinetic model (Michaelis–Menten’s equation). 3.2. pH-stat method Acetic acid (product of enzymatic hydrolysis of ATCH or ACH) decreases pH value of the reaction mixture. pH-stat (736 GP Titrino, Metrohm, Switzerland) continuous monitors pH value of the reaction mixture and at its fall automatic buret doses volumetric solution of KOH to the reaction mixture and pH value is adjusted to 8. The dependence of volume of KOH solution versus time is measured and this dependence is transformed to the dependence concentration of acetic acid [HA] versus t. From concentration of [HA] in reaction mixture versus time the kinetic model of hydrolysis ATCH can be evaluated.
388
Extended Abstracts / Chemico-Biological Interactions 157–158 (2005) 353–434
Fig. 1. pH-stat method.
4. Conclusions • It was found to be that hydrolyses of ATCH + BuCHE, ATCH + ACHE and ACH + BuCHE course from start to the exhaustion of substrate by Michaelis–Menten’s equation (Briggs–Haldane) for given conditions (pH, temperature) with irreversible decay of ES complex. • Average values of KM and Vm , rate constants ki and estimations of absolute concentration of given enzyme in reaction mixture [E]0 using non-linear regression model of GraphPad PRISM and solving of the system of differencial kinetic equations by GEPASI Program were found for single combinations of S and E. Agreement of proposed models and experiment is shown at Fig. 1. Demonstration of the agreement of theoretical kinetic model (1) and experimental data. Acknowledgements This work was financially supported by Czech Ministerium of Education, Helth and Sport—Research project CZ 340003/22/3340. doi:10.1016/j.cbi.2005.10.068
(24) Acetylcholinesterase mutants: oxime-assisted catalytic scavengers of organophosphonates Zrinka Kovarik a,b,∗ , Zoran Radi´c a, Vera Simeon-Rudolf b, Elsa Reiner b, Palmer Taylor a a
Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093-0636, USA b Institute for Medical Research and Occupational Health, Ksaverska cesta 2, P.O. Box 291, HR-10001 Zagreb, Croatia 1. Introduction Reaction of organophosphonates with acetylcholinesterase (AChE; EC 3.1.1.7) is characterized by the formation of serine-conjugated adducts that are only slowly reversible. Since oximes are the only known antidote to nerve agent poisoning that restore the activity of AChE, mutagenesis of AChE should enable one to develop more effective scavenging agents in which AChE mutants in combination with an oxime would complete a catalytic cycle of hydrolysis of the organophosphate by rapid inhibition followed by rapid nucleophile-mediated hydrolysis of the phosphonyl enzyme conjugate. We enlarged the active site gorge of mouse AChE with mutations Y337A, F295L and F297I. Based on rapid inhibition of mutants with SP -cycloheptyl methylphosphonyl thiocholine (SP -CHMPTCh) [1,2] and enhance∗
Corresponding author. Tel.: +385 1 46 73 188; fax: +385 1 46 73 303. E-mail address: [email protected] (Z. Kovarik).