Physical Evidence for the Existence of at Least Two Salt-dependent Forms of α-Chymotrypsin at Slightly Acid pH

Abstract

Abstract The interpretation of kinetic data for complex formation between the enzyme, α-chymotrypsin, and the substrate, methyl hippurate (described fully in the preceding paper), involves a change in conformation or structure of the enzyme when potassium chloride is added up to concentrations of 0.5 m; beyond this concentration it was suggested that no further change in the enzyme takes place. The physical evidence for what appears to be a conformational change is presented in this report. Continuous variation experiments using the refractive index and ultraviolet absorption as the measured parameters indicate an interaction between the enzyme and the salt; the nonlinear curves cross at 0.5 m ionic strength. Ultraviolet absorption at 282.5 mµ, optical rotation of the Cotton effect at 233 mµ, and the ellipticity at 231 mµ of the circular dichroism spectra of α-chymotrypsin change in a similar manner when potassium chloride is added. These values all increase up to an ionic strength of 0.5 m and then become independent of salt concentration, which correlates very well with the kinetic data. The helical content of the peptide chain appears to increase upon addition of potassium chloride. It is proposed that the binding area of the enzyme which is dependent on ion pairing between isoleucine 16 and aspartate 194 is that which is involved in hydrophobic binding of the aromatic moiety of the substrate. Some possible relationships of the findings to the known crystallographic structure of the enzyme are suggested.