HYDROPHOBIC INTERACTIONS CONTROL ZYMOGEN ACTIVATION IN THE TRYPSIN FAMILY OF SERINE PROTEASES

Trypsinogen is converted to trypsin by the removal of a peptide from t he N terminus, which permits formation of a salt bridge between the ne w N-terminal lie (residue 16) and Asp194. Formation of this salt bridg e triggers a conformational change in the ''activation domain'' of try psin, creating the SI binding site and oxyanion hole. Thus, the activa tion of trypsinogen appears to represent an example of protein folding driven by electrostatic interactions. The following trypsin mutants h ave been constructed to explore this problem: Asp194Asn, Ile16Val, Ile 16la, and Ile16Gly. The bovine pancreatic trypsin inhibitor (BPTI), be nzamidine, and leupeptin affinities and activity and pH-rate profiles of these mutants have been measured. The changes in BPTI and benzamidi ne affinity measure destabilization of the activation domain. These ex periments indicate that hydrophobic interactions of the Ile 16 side ch ain provide 5 kcal/mol of stabilization energy to the activation domai n while the salt bridge accounts for 3 kcal/mol, Thus, hydrophobic int eractions provide the majority of stabilization energy for the trypsin ogen to trypsin conversion. The pH-rate profiles of I16A and I16G are significantly different than the pH-rate profile of trypsin, further c onfirming that the activation domain has been destabilized. Moreover, these mutations decrease k(cat)/K-m and leupeptin affinity in parallel with the decrease in stability of the activation domain. Acylation is selectively decreased, while substrate binding and deacylation are no t affected, Together these observations indicate that the stability of protein structure is an important component of transition state stabi lization in enzyme catalysis. These results also suggest that active z ymogens can be created without providing a counterion for Asp194, and thus have important implications for the elucidation of the structural features which account for the zymogen activity of tissue plasminogen activator and urokinase.