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

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 degrees C-40 degrees C by spectrophotometry in TRIS buffer, pH 8.0, using both initial rate analysis and progress curve analysis. The kinetic parameters (catalytic constant k(cat); Michaelis constant K-m; product inhibition constant K-p) were determined by nonlinear regression. All parameters increased with temperature, but the ratios k(cat)/K-m and K-p/K-m 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. (C) 2013 Elsevier Masson SAS. All rights reserved.