Steady-State Kinetics and Thermodynamics of the Hydrolysis of β-Lactoglobulin by Trypsin

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Abstract
Hydrolysis of β-lactoglobulin (in an equimolar mixture of the A and B variant) by trypsin in neutral aqueous solution [pH 7.7 at 25 °C, ionic strength 0.08 (NaCl)] was followed by capillary electrophoresis and thermodynamic parameters derived from a Michaelis−Menten analysis of rate data obtained at 10, 20, 30, and 40 °C for disappearance of β-lactoglobulin. Enthalpy of substrate binding to the enzyme and the energy of activation for the catalytic process were found to have the values, ΔHbind = −28 ± 4 kJ mol-1 and Ea = 51 ± 18 kJ mol-1, respectively. Thus, β-lactoglobulin shows an enthalpy of activation for free substrate reacting with free enzyme of about 21 kJ mol-1, corresponding to a transition state stabilization of 60 kJ mol-1 when compared to acid-catalyzed hydrolysis. The catalytic efficiency of trypsin in hydrolysis of β-lactoglobulin is increased significantly by temperature; however, this effect is partly counteracted by a weaker substrate binding resulting in an increase by only 25%/10 °C in overall catalytic efficiency. Keywords: β-Lactoglobulin; trypsin; hydrolysis; steady-state kinetics; thermodynamics