Butyrylcholinesterase inhibition by ethopropazine enantiomers: evaluation of kinetic models (CROSBI ID 519434)
Prilog sa skupa u zborniku | sažetak izlaganja sa skupa | domaća recenzija
Podaci o odgovornosti
Šinko, Goran ; Stojan, Jure ; Goličnik, Marko ; Grubič, Zoran ; Kovarik, Zrinka ; Simeon-Rudolf Vera
engleski
Butyrylcholinesterase inhibition by ethopropazine enantiomers: evaluation of kinetic models
Butyrylcholinesterase (BChE, EC 3.1.1.8) is a serine hydrolase with a distinctively high catalytic turnover of acyl choline esters. The Michaelis-Menten model does not fully describe the hydrolysis of acetylthiocholine (ATCh) by BChE, because it involves only one enzyme-substrate complex. Therefore, we tested four kinetic models for ATCh hydrolysis by equine BChE in the presence and in the absence of a chiral reversible inhibitor ethopropazine (10-(2-diethyl-aminopropyl)phenothiazine hydrochloride). The kinetic models differed in the number and type of enzyme-substrate, enzyme-inhibitor and enzyme-substrate-inhibitor complexes, and in reversible and irreversible steps leading from one complex to another. To evaluate BChE’ s stereoselectivity the enzyme was inhibited by racemic and optically pure ethopropazine. Experiments were done using the Ellman's method at 12 °C on a stopped-flow spectrophotometer. All initial hydrolysis rates determined at various ATCh concentrations (2-75000 µ ; ; M) in the presence and in the absence of ethopropazine (0.5-10 µ ; ; M) were simultaneously fitted to equations derived from the kinetic models. The best model that resulted with the smallest sum of squares of deviations, about ten times smaller than with the Michaelis-Menten model, was one with six parameters for substrate hydrolysis and two parameters for enzyme inhibition. Additional criteria for evaluation of the models were the convergence of the fit and that standard deviation value does not exceed value of the pertinent constant. The dissociation constant of the peripherally bound substrate-BChE complex was 0.4 mM and of the peripherally bound substrate to the acetylated BChE 0.7 mM. The acylation rate constant was 4.8· ; ; ; ; ; 104 M-1min-1 and the deacylation rate constant 1.98· ; ; ; ; ; 108 min-1. Parameters that describe the influence of the peripherally bound substrate on acylation and deacylation, a and b, were 0.08 and 1.05, respectively. Dissociation constants of the BChE-ethopropazine complex were 16, 42 and 26 nM, for R-, S- and racemic ethopropazine, respectively, showing that the enzyme’ s affinity for R- was three times greater than for S-ethopropazine.
kinetic models; evaluation; ethopropazine; butyrylcholinesterase
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Podaci o prilogu
138-x.
2006.
objavljeno
Podaci o matičnoj publikaciji
Congress of the Croatian Society of the Biochemistry and Molecular Biology on the occasion of the 30th Anniversary with international participation, Book of Abstracts
Kovarik, Zrinka
Zagreb: Hrvatsko društvo za biokemiju i molekularnu biologiju (HDBMB)
Podaci o skupu
Congress of the Croatian Society of the Biochemistry and Molecular Biology on the occasion of the 30th Anniversary with international participation
poster
03.10.2006-07.10.2006
Vodice, Hrvatska