Naslov
Butyrylcholinesterase inhibition by ethopropazine enantiomers: evaluation of kinetic models

Autori
Šinko, Goran ; Stojan, Jure ; Goličnik, Marko ; Grubič, Zoran ; Kovarik, Zrinka ; Simeon-Rudolf Vera

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
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), 2006

Skup
Congress of the Croatian Society of the Biochemistry and Molecular Biology on the occasion of the 30th Anniversary with international participation

Mjesto i datum
Vodice, Hrvatska, 03.10.2006. - 07.10.2006

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
kinetic models; evaluation; ethopropazine; butyrylcholinesterase

Sažetak
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 &micro ; ; M) in the presence and in the absence of ethopropazine (0.5-10 &micro ; ; 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&middot ; ; ; ; ; 104 M-1min-1 and the deacylation rate constant 1.98&middot ; ; ; ; ; 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.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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