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Kinetics of Hydrolysis of Acetylthiocholine and Acetylcholine by Cholinesterases
Authors: Komersová Alena | Komers Karel | Zdražilová Pavla
Year: 2004
Type of publication: ostatní - přednáška nebo poster
Name of source: VIII. International Meeting on Cholinesterases
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Language Name Abstract Keywords
cze Kinetika hydrolýzy acetylthiocholinu a acetylcholinu cholinesterázami. Acetylthiocholin (ATCH) se používá k testování enzymatické hydrolýzy acetylcholinu (ACH) in vitro, protože mechanismus hydrolýzy ATCH je kvalitativně podobný hydrolýze ACH a průběh reakce lze kvantitativně on-line sledovat dvěma nezávislými metodami: spektrofotometricky (stanovení thiocholinu, produktu hydrolýzy ATCH, Ellmanovou metodou) a elektrochemicky (stanovení kyseliny octové - produkt hydrolýzy ATCH -pH statovou metodou). Ellmanova metoda byla použita ke sledování mechanismu enzymatické hydrolýzy ATCH pomocí butyrylcholinesterázy (BCHE) a acetylcholinesterázy (ACHE). Všechny zjištěné závislosti splňují až do totálního vyčerpání substrátu kinetiku Michaelis-Mentenové s druhým irreverzibilním krokem. Vyhodnocení bylo prováděno na základě diferenciálního a integrálního tvaru kinetického modelu Michaelis-Mentenové. Pro každý szstém byly stanoveny optimální hodnoty Michaelisovy konstanty (KM), maximální reakční rychlost (Vm), kinetické konstanty jednotlivých reakčních kroků a absolutní koncentrace enzymu. enzyme, inhibitors, kinetics, hydrolysis, acetycholinesterase
eng Kinetics of Hydrolysis of Acetylthiocholine and Acetylcholine by Cholinesterases Acetylthiocholine (ATCH) is used for testing of enzymatic hydrolysis of acetylcholine (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). Ellman´s method was used for testing of the mechanism of enzymatic hydrolysis of ATCH by butyrylcholinesterase (BCHE) and acetylcholinesterase (ACHE). In these experiments the dependences of thiocholine concentration vs. time at 25oC and pH 8 were measured. These dependences correspond to the Michaelis-Menten´s equation. It was found that all hydrolyses confirm this equation with the second irreversible step up to the total exhaustion of the substrate ATCH. 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. The obtained results are now used in the research of inhibition of the enzymatic hydrolyses of ATCH and ACH by BCHE and ACHE with a group of new inhibitors ? carbamates and carbazates. The best inhibitors will be tested with native preparates and suggested to the clinical tests as potential drugs of Alzheimer disease. enzyme, inhibitors, kinetics, hydrolysis, acetycholineste

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