Characterization of a control switch in chymotrypsin.

Kinetic and thermodynamic parameters of the conformational change accompanying the binding to chymotrypsin of a substance carrying substrate specificity have been determined by a chemical relaxation method. An analysis of the concentration dependence of the two relaxation times and the associated amplitudes permitted the evaluation of rat constants, reaction enthalpies, and entropies of the individual steps. The pH and temperature dependencies of these parameters allowed, together with the known topography of the molecule, the identification of the ionizing groups involved in the regulation of the two steps. The combined evidence was used to compose a detailed mechanism for the function of the conformational change. The hypothesis is proposed that the side chain of ASp-194 is acting as the tongue of a switch which is moving between the positively charged His-57 side chain (catalytically inactive state) and the positively charged alpha-amino group of Ile-16 (active state), thus activating or deactivating the catalytic site. This control switch is very sensitive to changes in pH and temperature.