Steady-state kinetics and thermodynamics of the hydrolysis of beta-lactoglobulin by trypsin.

Hydrolysis of beta-lactoglobulin (in an equimolar mixture of the A and B variant) by trypsin in neutral aqueous solution [pH 7.7 at 25 degrees 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 degrees C for disappearance of beta-lactoglobulin. Enthalpy of substrate binding to the enzyme and the energy of activation for the catalytic process were found to have the values, DeltaH(bind) = -28 +/- 4 kJ mol(-)(1) and E(a) = 51 +/- 18 kJ mol(-)(1), respectively. Thus, beta-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 beta-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 degrees C in overall catalytic efficiency.