engleski

Kinetics of interaction of ethopropazine enatiomers with butyrylchopolinesterase and acetylcholinesterase

The association and dissociation rates of (+)ethopropazine and (-) ethopropazine with wild-type mouse and horse butyrylcholinesterases (BChE), and mutant mouse acetylcholinesterase (AChE) were studied in order to analyze elements of stereo selectivity in two similar but distinct enzyme templates. Reaction traces at 23 oC in 0.1 M phosphate buffer pH 7.0 were recorded in millisecond time frame using Applied Photophysics stopped-flow apparatus equipped with fluorescence detection. Dissociation rate constants evaluated for BChEs were three to four-fold faster for (-)enantiomer (130 (1/min) vs. 30 (1/min) for horse BChE) , while association rate constants for enantiomers were similar (1.4 (1/nM*min) vs. 1.0 (1/nM*min) for horse BChE), resulting in lower equilibrium dissociation constant and better binding of (+) ethopropazine with BChE. In the AChE template the preferential binding of (+)ethopropazine was observed with Tyr337Ala mutant, but with an order of magnitude greater stereo specificity. Binding preference for Tyr124Gln AChE mutant, however, was inversed and (-)ethopropazine (Kd of about 1.8 uM ) bound several fold better than (+)enantiomer. Inhibition of enzymic acetylthiocholine hydrolysis by ethopropazine enantiomers yielded equilibrium inhibition constants similar to equilibrium dissociation constants derived from stopped-flow rate measurements. In conclusion, active center gorge of AChE, lined with larger number of aromatic residues than the gorge of BChE, provides narrower and more stereo selective environment for binding of ethopropazine.

butyrylcholinesterase; acetylcholinesterase; ethopropazine; binding sites

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