engleski

Evaluation of butyrylcholinesterase stereoselectivity in interaction with enantiomers of ethopropazine

Stereoselectivity of biological macromolecules originates from chiral amino acids which are building blocks for enzymes and other proteins. It is usual for enzyme to show stereoselectivity in interaction with chiral substrate or inhibitor. We studied butyrylcholinesterase (BChE, EC 3.1.1.8) stereoselectivity in interaction with enantiomers of ethopropazine. BChE is related to acetylcholinesterase (AChE, EC 3.1.1.7) which is involved in neurotransmission and they share 54% of sequence homology. BChE is involved in hydrolysis of various esters and xenobiotics which makes it useful in prodrug conversion, i.e. bambuterol used in the treatment of asthma. Ethopropazine is a chiral drug used in treatment of Parkinson’s disease in form of a racemate, an equimolar mixture of individual enantiomers. We performed a series of BChE activity measurements in which we used low, mid and high substrate concentrations (100 µM, 1 mM and 100 mM) and performed BChE inhibition with addition of racemic and enantiomeric pure ethopropazine into the reaction mixture to evaluate BChE stereoselectivity. We evaluated stereoselectivity of a free enzyme and different enzyme-substrate complexes by using various substrate concentrations. Different BChE-substrate complexes occur at high substrate concentration well above KM value for acetylthiocholine (0.79 mM, 25°C). It is known that temperature may have a significant effect on enzyme structure dynamics. To test link between BChE structure dynamics and stereoselectivity we performed activity measurements in the absence and presence of ethopropazine at 12, 20, 25, 30 and 37 °C. Our results will give more insight into BChE stereoselectivity with purpose of future drug design especially for chiral drugs or prodrugs.

temperature effect on enzyme structure dynamics

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