Effect of a water-miscible organic solvent on the kinetic and structural properties of trypsin

Abstract

The effects of a water-miscible organic solvent (9:1 mix of DMF and DMSO) on the esterase and amidase kinetic parameters of trypsin have been investigated. The solvent selectively reduced the k cat for amidase activity relative to that for esterase activity. For both reaction types, K m increased sharply with increasing solvent concentration. Kinetic studies were complemented by fluorescent measurements of the enzyme's overall conformation and by EPR spectroscopy studies of the active-site microstructure. The decline in k cat for amidase activity began at solvent concentrations greater than 10%; this decrease correlated well with minor changes in the structure of the active site as indicated by EPR spectroscopy. In contrast, the esterase k cat showed no significant decrease throughout the solvent concentration range studied. The k cat values were based on the number of active sites as indicated by active site titration.