On Dielectric Constant and Enzymatic Kinetics

Abstract

The dielectric effects on trypsin and a-chymotrypsin activities have revealed that at pH 7.8 the active species of the former is the cation while that of the latter is the anion. The present study on the dielectric effects along the pH-activity curves shows that trypsin remains positive within the pH range of 5.5 to 8.5. Conversely, a-chymotrypsin is positive from pH 5.5 to 6.6, negative from 6.6 to about 8.1, and at pH 8.25 becomes positive again. The first point of inversion in charge sign shifts from 6.6 to 7.15 with the addition of 0.05 M phosphate buffer. The point of inversion does not seem to be modified significantly by changes in the substrate structure. At pH values near the point of inversion the plots of rate log vs. 100/D are broken lines formed by various straight portions, the slope of each varying progressively from a maximum positive to a maximum negative value. This suggests an effect of resonance possibly attributable to an imidazole group. As an attempt to explain the two observed points of sign inversion in a-chymotrypsin, the possibility is suggested that different enzyme configurations are disclosed by the combined action of pH and dielectric constant. On this theoretical basis, it is feasible that more than one isoionic point exists. I N T R O D U C T I O NIt was reported previously that the rate of ester hydrolysis by trypsin (5) or a-chymotrypsin (6) is modified by changes in the medium dielectric strength. By comparison with non-enzymatic reactions, it might be inferred from the behavior of these two enzymes that at p H 7.8 the active f o r m of trypsin is the cation and that of ot-chymotrypsin is the anion. These observations are in harmony with two earlier findings : (a) Northrop (16) deduced, by D o n n a n equilibrium considerations, that trypsin behaves like a univalent positive

On Dielectric Constant and Enzymatic Kinetics

“…The present observations and earlier ones (5,6) concerning the behavior of the trypsin-and a-chymotrypsin-catalyzed hydrolyses of BAEE show that the trend of dielectric and salt effects is determined principally by the enzyme charges.…”

“…In the case of urea, the dielectric constant was increased only from 78.5 to 88.5 (3.? M urea), because greater concentrations produce a rapid fall of enzyme activity (5). It can be observed in both Figs.…”

“…Substrate concentration, 0.008 M. Enzyme concentration, 10 #g per ml. p H range of 5.5 to 8.5, while a-chymotrypsin appears positive from p H 5.5 to about 6.6 and then changes to negative (5). A further study on the effect of ionic strength on the kinetics of these two enzymes (6) added support to the assumption that the charges involved in the activity of trypsin and a-chymotrypsin are different.…”