The activity of porcine pancreatic phospholipase A2 in 20% alcohol/aqueous solvent, by experiment and electrostatics calculations

The activity of porcine pancreatic phospholipase A2 (pla2), measured at pH 8, is reduced when methanol or ethanol is added to the aqueous solution. Finite difference electrostatics calculations were used to study the effect of modelling mixed solvents on the pK(a)s of histidine 48 and the amino-terminal group, both of which influence the pH-dependence of catalysis. Calculations and experiment indicate that these pK(a) values cannot account for the activity reduction. Charge separation in the transition state is destabilized in 20% alcohol solvent relative to 100% aqueous solvent. The calculated values, which are combinations of stabilizing and destabilizing contributions, are in qualitative agreement with experiment. Saturating dielectric theory is used to model solvent water ordering in a high electric field, and water dielectric structure is assumed to dominate at the 20% alcohol level. The observed agreement demonstrates the utility of transition state stabilization theory and continuum solvent modelling. It is further suggested that electrostatic effects on k(cat) contribute to the pH-dependence of activity around pH 7, and to previously reported activity changes for charge mutants.