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Kinetics of Hydrolysis of Acetylthiocholine by Cholinesterases
Authors: Zdražilová Pavla | Komers Karel | Štěpánková Šárka | Komersová Alena
Year: 2004
Type of publication: ostatní - přednáška nebo poster
Name of source: The 2004 Youger European Chemists´ Conference
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Language Name Abstract Keywords
cze Kinetika hydrolýzy acetylthiocholinu cholinesterázami Kinetika enzymatické hydrolýzy acetylthiocholinu (ATCH) pomocí butyrylcholinesterázy (BCHE) a acetylcholinesterázy (ACHE) byla studována pomocí dvou nezávislých metod, spektrální Ellmanovy (stanovení vznikajícího thiocholinu) a pH-statové (stanovení vznikající kyseliny octové) při 25oC a pH 8. Získané závislosti koncentrací uvedených produktů na čase, měřené až do vyčerpání ATCH byly porovnány s kinetickým modelem Michaelis-Mentenové s nevratným a současným vznikem obou produktů. Simulace experimentů byla provedena několika matematickými postupy. Bylo zjištěno, že uvedený kinetický model vyhovuje v celém průběhu reakce a byly vypočteny optimální hodnoty KM a Vm, rychlostních konstant k1, k-1 a k2, jakož i odhad absolutní výchozí koncentrace ezymu v reakční směsi. Enzyme, hydrolysis, inhibitors, acetylthiocholine, cholinesterases
eng Kinetics of Hydrolysis of Acetylthiocholine by Cholinesterases Acetylthiocholine (ATCH) is used for testing of enzymatic hydrolysis of acetylcholine (ACH) in vitro, because the mechanism of the hydrolysis is qualitatively similar to ACH and its reaction course can be quantitatively measured on-line by two independent methods: spectrophotometric (the Ellman´s method; determination of product thiocholine) and electrochemical (pH-stat method; determination of the product acetic acid). We used the Ellman´s method for testing the mechanism of enzymatic hydrolysis of ATCH by butyrylcholinesterase (BCHE) and acetylcholinesterase (ACHE). In these experiments the dependencies of concentrations of thiocholine vs. time at 25oC and pH 8 were measured. These dependencies were correlated with the validity of the Michaelis-Menten´s equation. It was found that with high probability, that all hydrolyses confirm this equation with the second irreversible step up to the total exhaustion of ATCH substrate. The correlations were made by means of differential and integral kinetic equations describing the given Michaelis-Menten model. In every experiment there were calculated optimal values of the Michaelis constant (KM), the maximum velocity (Vm), kinetic constants of single reaction steps and the absolute concentration of the used enzyme. The obtained results are now used in the research of inhibition of enzymatic hydrolyses of ATCH and ACH by BCHE and ACHE with a group of new inhibitors ? carbamates and carbazates. The ideal goal of this research is to find a new potential drug against the Alzheimer´s disease. Enzyme, hydrolysis, inhibitors, acetylthiocholine, cholinesterases

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