Calorimetric Characterization of Ligand Binding on Trypsinogen and Chymotrypsinogen A, with Evidence for Two Binding Sites on &hymotrypsin*

Heat is evolved when solutions of trypsinogen or chymotrypsinogen A are mixed with solutions of the amide or methyl ester derivatives of NQ-p-toluenesulfonyl-L-arginine, appropriate corrections being made for heats of reactant dilution. Exothermic reaction was also observed when the two ligands were mixed with tr-chymotrypsin; all measurements were made at 25”, pH 5.0. The dependence of apparent reaction heat upon ligand concentration was used to evaluate ligand binding site models, thereby providing values for changes in free energy and entropy as well. Binding to the zymogens was represented by a model having a single class of sites, but description of the ester-cr-chymotrypsin data required the postulation of two classes of ligand binding sites. Comparison with previous kinetic investigations leads to the view that the ligand binding site of the zymogen persists in the active enzyme, in addition to the binding site made available upon activation of the zymogen by trypsin.