Kinetic investigation of human dipeptidyl peptidase II (DPPII)-mediated hydrolysis of dipeptide derivatives and its identification as quiescent cell proline dipeptidase (QPP)/dipeptidyl peptidase 7 (DPP7)

The presence of DPPII (dipeptidyl peptidase 11; E.C. 3.4.14.2) has been demonstrated in various mammalian tissues. However, a profound molecular and catalytic characterization, including substrate selectivity, kinetics and pH-dependence, has not been conducted. In the present study, DPPII was purified from human seminal plasma to apparent homogeneity with a high yield (40 %) purification scheme, including an inhibitor-based affinity chromatographic step. The inhibitor lysyl-piperidide (K-i similar to 0.9 mu M at pH 5.5) was chosen, as it provided a favourable affinity/recovery ratio. The human enzyme appeared as a 120 kDa homodimer. Mass spectrometric analysis after tryptic digestion together with a kinetic comparison indicate strongly its identity with QPP (quiescent cell proline dipeptidase), also called dipeptidyl peptidase 7. pH profiles of both k(cat) and k(cat)/K-m clearly demonstrated that DPPII/QPP possesses an acidic and not a neutral optimum as was reported for QPP. Kinetic parameters of the human natural DPPII for dipeptide-derived chromogenic [pNA (p-nitroanilide)] and flu-orogenic [4Me2NA (4-methoxy-2-naphthylamide)] substrates were determined under different assay conditions. DPPII preferred the chromogenic pNA-derived substrates over the fluorogenic 4Me2NA-derived substrates. Natural human DPPII showed high efficiency towards synthetic substrates containing proline at the P, position and lysine at P-2. The importance of the P-1(') group for P-2 and P-1 selectivity was revealed, explaining many discrepancies in the literature. Furthermore, substrate preferences of human DPPII and dipeptidyl peptidase IV were compared based on their selectivity constants (k(cat/)K(m)). Lys-Pro-pNA (k(cat)/ K-m 4.1 x 10(6) s(-1) (.) M-1) and Ala-Pro-pNA (k(cat)/K-m 2.6 x 10(6) s(-1) M-1) were found to be the most sensitive chromogenic substrates for human DPPII, but were less selective than Lys-Ala-pNA (k(cat/)K(m) 0.4 x 10(6) s(-1) (.) M-1).