Conformational states of chymotrypsin at high pH: Temperature effects on catalysis and binding

Abstract

Studies on the effects of temperature on the chymotrypsin-catalyzed hydrolysis of specific and nonspecific substrates reveal markedly nonlinear Arrhenius and van't Hoff plots. The investigations include the δ- and α-forms of the enzyme at pH 7.8–9.6 and 0–40 °C. Rigorous error-analysis of the data by computer indicates that these nonlinear responses are not artifactual. By determining the effects of temperature and pH on the relative rates of the acylation and deacylation processes, little or no evidence is found for the nonlinear plots being caused by a change in rate-limiting step as these parameters vary. Model calculations also show that van't Hoff plots that are concave downward, as frequently observed here, cannot be produced by a mechanism involving a change in rate-limiting step. A variety of extra-kinetic data support the view that conformation changes in the enzyme are the most likely explanation. Detailed comparisons of activation parameters calculated for each enzyme-substrate interaction show clearly that changes in enthalpy values are compensated for by changes in entropy. Relative to the α-enzyme, δ-chymotrypsin maintains an active-site conformation more favorable for catalysis over a wider temperature range. Different activation parameters dominate the catalytic process with specific substrates, as compared to nonspecific ones. At high pH the binding process is no longer dominated by apolar interactions even with specific substrates, and the enzyme tends to lose its specificity. Overall, the data indicate that the selectivity and, by inference, the structural integrity of the active site decrease as one proceeds from low to high temperature, from neutral to alkaline pH, from δ- to α-chymotrypsin, or from specific to nonspecific amino acid sidechain groups. These studies also have implications for conclusions drawn from data collected at temperatures other than 37 °C.