Dipeptidyl peptidase I: importance of progranzyme activation sequences, other dipeptide sequences, and the N-terminal amino group of synthetic substrates for enzyme activity

The broadly reactive cysteine protease dipeptidyl peptidase I (DPPI, cathepsin C) is thought to activate all progranzymes (zymogens of lymphocyte serine proteases) to form mature granzymes. We synthesized dipeptide 7-amino-4-methylcoumarin (AMC) substrates containing progranzyme activation sequences and showed that they were efficiently hydrolyzed by DPPI. However, DPPI will not hydrolyze Ile-Ile-AMC, the N-terminal dipeptide sequence found in mature granzymes. Introduction of the nonphysiological homophenylalanine (Hph) residue at P1 resulted in the best substrate Ala-Hph-AMC for DPPI (kcat/Km=9,000,000 M−1 s−1). The charged N-terminal amino group of the substrate was essential and replacement of the NH2 group with OH or NH(CH3) in Gly-Phe-AMC reduced the kcat/Km value by two to three orders of magnitude. A hydrazide azaglycine analog, NH2NHCO-Phe-AMC, was not hydrolyzed at pH 5.5, but underwent slow hydrolysis at lower pHs where the amino group is partially protonated. DPPI also failed to hydrolyze NH2COCH2-Phe-AMC, where the NH2 group is unprotonated. The results reported in this paper should be useful in the design of better DPPI inhibitors to block granzyme maturation and granzyme-dependent apoptosis.