Osmotic pressure effects on EcoRV cleavage and binding

Investigations of DNA binding proteins frequently measure pH and salt depen dence, but relatively few studies measure protein binding in high concentra tions of small molecules often found in vivo. We have measured kinetics of the restriction enzyme EcoRV in concentrated solutions of three small cosol vents that produce osmotic pressures from 0.25 to 2.5 mol/kg (6 to 62 atm o r water activity of 0.995 to 0.956). We have correlated DNA cleavage and bi nding parameters with four solution parameters (dielectric constant, viscos ity, water concentration, and water activity). We found that the responses of maximum velocity (V-max) and the dissociation constant for nonspecific b inding (K-dms) best correlate with water activity. The Michaelis constant ( K-m) correlates with both water activity and solution viscosity, the latter due to the highly dilute reactant concentrations, which make enzyme-substr ate combination diffusion limited. Dielectric constant does not influence a ny of the kinetic parameters, which is consistent with a view that protein and DNA are preferentially hydrated, and excluded solutes cannot affect the local dielectric constant.