engleski

Horse butyrylcholinesterase inhibition with ethopropazine enantiomers : Temperature influence on stereoselectivity

Reversible inhibition of horse serum butyrylcholinesterase (BChE, EC 3.1.1.8) with ethopropazine (10 ­(2 -diethylaminopropyl) phenothiazine hydrochloride) enantiomers was studied at 12, 25 and 37 °C. Enzyme activity was measured with acetylthiocholine as substrate. The binding constants of ethopropazine and BChE evaluated from the degree of enzyme inhibition as a function of the substrate concentration (0.1 - 3.0 mM) were: 3.7, 2.1 and 0.9 mM(-1) for the S and 9.2, 5.3 and 1.8 mM(-1) for R enantiomer, at 12, 25 and 37 °C, respectively. The standard enthalpies of enantiomer binding to BChE were ­40 and ­48 kJ/mol for S and R ethopropazine, respectively. The binding of the R enantiomer seems more exothermic by 8 kJ/mol , i.e. more favourable. Stereoselectivity, the ratio of binding constants at the same temperature, was 2.5 at both 12 °C and 25 °C and 2.0 at 37 °C. By increasing the temperature from 12 °C to 25°C, binding constants decreased 1.7fold, and stereoselectivi ty remained the same although affinity was lower. From inhibition experiments, the enzyme ­substrate dissociation constants K(s) were also derived. At 12 and 25°C the same K(s) values were obtained with both enantiomers: 0.6 mM at 12°C and 1.2 mM at 25°C. At 37°C, K(s) values were 3.7 and 5.3 mM from the experiments with S and R ethopropazine, respectively. A drop in stereoselectivity at 37 °C indicates a change in enzyme ­inhibitor interactions. The binding of enantiomers to BChE seems to be enthalpy driven, which means that stereoselectivity increases with decreasing temperature.

butyrylcholinesterase; stereoselectivity; ethopropazine; temperature

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