2. Author's Information
    Marie-Berthe MAES
    Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein1, B-2610 Antwerp, Belgium

    Anne-Marie LAMBEIR
    Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein1, B-2610 Antwerp, Belgium

    Kambiz GILANY
    Laboratory of Protein Chemistry, Department of Biomedical Sciences, University of Antwerp, Universiteitsplein1, B-2610 Antwerp, Belgium

    Kristel SENTEN
    Laboratory of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein1, B-2610 Antwerp, Belgium

    Pieter VAN der VEKEN
    Laboratory of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein1, B-2610 Antwerp, Belgium

    Barbara LEITING
    Department of Metabolic Disorders, Merck Research Laboratories, Mail code RY50G-236, P.O. Box 2000, Rahway, NJ 07065, U.S.A.

    Koen AUGUSTYNS
    Laboratory of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein1, B-2610 Antwerp, Belgium

    Simon SCHARPÉ
    Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein1, B-2610 Antwerp, Belgium

    Ingrid DE MEESTER
    Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein1, B-2610 Antwerp, Belgium
  3. Abstract
    The presence of DPPII (dipeptidyl peptidase II; 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 (Ki~0.9 μM 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 kcat and kcat/Km 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 fluorogenic [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 P1 position and lysine at P2. The importance of the P1′ group for P2 and P1 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 (kcat/Km). Lys-Pro-pNA (kcat/Km 4.1×106 s−1·M−1) and Ala-Pro-pNA (kcat/Km 2.6×106 s−1· M−1) were found to be the most sensitive chromogenic substrates for human DPPII, but were less selective than Lys-Ala-pNA (kcat/Km 0.4×106 s−1·M−1).
    Keywords
    dipeptidyl peptidase (DPP), kinetics, pH profile, proline, quiescent cell proline dipeptidase (QPP)
    DOI: 10.1042/BJ20041156
    ADLID: 36448-v4
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  1. Keywords
    dipeptidyl peptidase (DPP) kinetics pH profile proline quiescent cell proline dipeptidase (QPP)
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