Distinct Properties of Prohormone Thiol Protease (PTP) Compared to Cathepsins B, L, and H: Evidence for PTP as a Novel Cysteine Protease

Abstract

The prohormone thiol protease (PTP) has been demonstrated as a major processing enzyme involved in converting the enkephalin precursor to active opiate enkephalin peptides. In this report, PTP was distinguished from other mammalian cysteine proteases, cathepsins B, L, and H, with regard to selectivity for monobasic and paired basic residue-containing peptide-MCA substrates, particular types of proteolytic activities, and sensitivity to active-site-directed peptide inhibitors. PTP cleaved at both COOH- and NH 2-terminal sides of basic residues within peptide-MCA substrates containing monobasic and dibasic sites. PTP showed greatest activity with Z-Phe-Arg-MCA and Bz-Val-Leu-Lys-MCA, and lower levels of activity with peptide substrates containing the paired basic residues Arg-Arg, Lys-Arg, and Lys-Lys. Cathepsins B and L also cleaved monobasic and paired basic residues at both COOH- and NH 2-terminal sides of basic residue(s), but differed from PTP in their preference for the same series of peptide-MCA substrates. Cathepsin H appeared to prefer cleaving at the NH 2-terminal side of basic residues. PTP showed no aminopeptidase activity which is known for cathepsin H. Also, PTP does not exhibit dipeptidylcarboxypeptidase activity, a property of cathepsin B; PTP demonstrates no elastinolytic activity, a characteristic of cathepsin L. Importantly, the sensitivity of PTP to active-site-directed peptide diazomethane inhibitors and E-64c differs from that of cathepsins B, L, and H. E-64c, a selective cysteine protease inhibitor, was the most effective inhibitor of PTP with a second-order rate constant of inactivation, k 2, of 6,710,000 M −1 s −1. These biochemical properties of PTP distinguish it from cathepsins B, L, and H, providing further support for PTP as a novel member of the family of cysteine proteases.