title: Osmotic pressure effects on EcoRV cleavage and binding.
abstract: Investigations of DNA binding proteins frequently measure pH and salt dependence, but relatively few studies measure protein binding in high concentrations of small molecules often found in vivo. We have measured kinetics of the restriction enzyme EcoRV in concentrated solutions of three small cosolvents that produce osmotic pressures from 0.25 to 2.5 mol/kg (6 to 62 atm or water activity of 0.995 to 0.956). We have correlated DNA cleavage and binding parameters with four solution parameters (dielectric constant, viscosity, water concentration, and water activity). We found that the responses of maximum velocity (Vmax) and the dissociation constant for nonspecific binding (Kd,ns) best correlate with water activity. The Michaelis constant (Km) correlates with both water activity and solution viscosity, the latter due to the highly dilute reactant concentrations, which make enzyme-substrate combination diffusion limited. Dielectric constant does not influence any 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.
authors.affiliation: Department of Biochemistry, University of Minnesota, St. Paul 55108, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Wenner
authors.forename: V A
authors.initials: VA
authors.lastname: Bloomfield
mesh_terms: D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009997:Osmotic Pressure; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000378:metabolism*; D012997:Solvents; D014783:Viscosity
chemical_list: D012997:Solvents; C056127:DNA modification methylase EcoRV; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)