Determinants of chymotrypsin C cleavage specificity in the calcium‐binding loop of human cationic trypsinogen

The pancreatic serine protease chymotrypsin  C ( CTRC ) cleaves the L eu81– G lu82 peptide bond in the calcium‐binding loop of human cationic trypsinogen and thereby promotes its degradation. This serves as a protective mechanism against ectopic trypsinogen activation in the pancreas. In the present study, we demonstrate that cleavage of the Leu81–Glu82 peptide bond by CTRC is highly specific, and other human pancreatic chymotrypsins ( CTRB 1, CTRB 2 and CTRL 1) and elastases ( ELA 2A, ELA 3A and ELA 3 B ) do not catalyze this reaction. To elucidate the mechanistic basis for CTRC specificity, we surveyed the primary ( P 1) cleavage preference of these pancreatic proteases on peptide substrates. We found that CTRC cleaved after a P 1 L eu with at least tenfold higher catalytic efficiency than other enzymes tested. To assess extended sub‐site interactions, we introduced A la mutations into human cationic trypsinogen at the P 3, P 1′ P 3′ and P 4′ amino acid positions, where P 1– P 1′ corresponds to L eu81– G lu82. Interestingly, CTRC ‐mediated cleavage was stimulated threefold by mutation E 82 A and unaffected by mutations E 79 A and N 84 A , but all three mutations compromised specificity and resulted in increased cleavage by ELA 2 A . Mutation E 85 A decreased CTRC cleavage by twofold. Remarkably, other chymotrypsins and elastases did not cleave human cationic trypsinogen even with the L 81 F or L81A mutations, which introduced favorable P 1 residues for these enzymes. We conclude that specific cleavage of the Leu81–Glu82 peptide bond in human cationic trypsinogen by CTRC is primarily determined by its distinctively high activity on leucyl peptide bonds, with the P 1′ G lu82, P 3′ A sn84 and P 4′ G lu85 residues serving as additional specificity determinants.