Abstract
The digestive protease chymotrypsin (CTR) protects the pancreas against harmful trypsin activity by promoting degradation of trypsinogen. Recently, we demonstrated that Arg236 is responsible for the higher proteolytic activity and better trypsinogen degrading capability of human CTRB2 compared to CTRB1. Introduction of Arg236 into CTRB1, which normally carries Asp236, dramatically increased degradation of human anionic trypsinogen. Here, we explored whether we could improve the activity of mouse CTRB1 by changing Gly236 to Arg (G236R mutant) and/or by widening the substrate binding pocket (A244G mutant). We found that mutant G236R cleaved mouse anionic (T8) trypsinogen at Phe150 with 32-fold improved efficiency. In contrast, mutant G236R digested mouse cationic (T7) trypsinogen and bovine beta-casein at the same rate as wild-type mouse CTRB1. Mutation A244G reduced the activity of mouse CTRB1 against the two trypsinogen isoforms and casein. Double-mutant G236R-A244G cleaved mouse anionic (T8) trypsinogen 9.8-fold better than wild-type CTRB1 but 3.3-fold slower than single mutant G236R. Mutant G236R-A244G digested mouse cationic (T7) trypsinogen at the same rate as single-mutant A244G but degraded casein 2.3-fold slower. Taken together, the observations indicate that in the context of mouse CTRB1 the Arg236 residue increases protease activity in a substrate-specific manner, while Gly244 has an overall negative impact. The results will inform the design of preclinical mouse models with higher trypsinogen degradation ability and enhanced resilience against pancreatitis.