Abstract
The zymogen and mature enzyme forms of trypsin-like serine proteases exhibit a wide range of activities. The prototypical trypsinogen−trypsin system is an example of a minimally active zymogen and a maximally active mature protease. The present work identifies several features of trypsinogen which govern its activity. Our results indicate that rat trypsin is 108-fold more active than rat trypsinogen. Rat trypsinogen appears to be less active than bovine trypsinogen. His40 is believed to be an important determinant of zymogen activity. We are unable to verify this role for His40 in trypsinogen since the mutation of His40 to Phe appears to change the trypsin−substrate interface. Deletion of the N-terminal Ile16 from trypsin is expected to produce a trypsinogen-like protein since the Ile16−Asp194 salt bridge cannot form. Such mutants have higher activity and BPTI affinity than trypsinogen, which indicates that the activation peptide stabilizes the inactive trypsinogen conformation. The mutation of Lys15 to Ala increases the BPTI affinity and activity of trypsinogen to an even greater extent; thus, removal of Lys15 can account for the effect of the loss of the activation peptide. These results suggest that Lys15 is an important determinant of zymogen activity. The mutation of Asp194 to Asn also increases the BPTI affinity and activity of trypsinogen. This result suggests that in addition to stabilizing the active conformation of trypsin via the Ile16−Asp194 salt bridge, Asp194 also maintains the inactive conformation of trypsinogen. A correlation exists between the values of k cat/K m and BPTI affinity of mutant trypsinogens and trypsins. However, the slope of this correlation is 0.64, which indicates that different “active” conformations are involved in BPTI binding and substrate hydrolysis. ΔI16V17 trypsinogen is the lone outlier; its BPTI affinity is higher than would be expected based on the value of k cat/K m. We show that the rate of BPTI association is slower for ΔI16V17 trypsinogen than for a mutant trypsinogen with a similar BPTI affinity. This observation suggests that BPTI binds to an “active” trypsinogen conformation that is not kinetically accessible to substrates.