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Temperature dependence of binding and catalysis for human serum arylesterase/paraoxonase.

Abstract : : The influence of temperature upon the hydrolysis of phenyl acetate, catalysed by purified human serum arylesterase/paraoxonase (E. C. 3.1.8.1), was studied in the temperature range 10 °C-40 °C by spectrophotometry in TRIS buffer, pH 8.0, using both initial rate analysis and progress curve analysis. The kinetic parameters (catalytic constant kcat; Michaelis constant Km; product inhibition constant Kp) were determined by nonlinear regression. All parameters increased with temperature, but the ratios kcat/Km and Kp/Km remained practically constant. Binding of both substrate and reaction product (phenol) was exothermic. A negative entropic term accounted for about 50% of the enthalpy change for both the binding and catalytic steps. Thermodynamic analysis suggested that: (1) the rate-limiting step is the nucleophilic attack of the carbonyl group of the substrate by a water molecule, (2) the active site is preorganized with no induced fit, (3) the enzyme-bound calcium plays an important role in stabilizing both the substrate and the transition state. The practical implications of these results are discussed.
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Dernière modification le : jeudi 5 mars 2020 - 17:57:58
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Jean Debord, Jean-Claude Bollinger, Michel Harel, Thierry Dantoine. Temperature dependence of binding and catalysis for human serum arylesterase/paraoxonase.. Biochimie, Elsevier, 2014, 97, pp.72-77. ⟨10.1016/j.biochi.2013.09.022⟩. ⟨hal-00875973⟩

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