title: Investigation of the dimer interface and substrate specificity of prolyl dipeptidase DPP8.
abstract: DPP8 belongs to the family of prolyl dipeptidases, which are capable of cleaving the peptide bond after a penultimate proline residue. Unlike DPP-IV, a drug target for type II diabetes, no information is available on the crystal structure of DPP8, the regulation of its enzymatic activity, or its substrate specificity. In this study, using analytical ultracentrifugation and native gel electrophoresis, we show that the DPP8 protein is predominantly dimeric when purified or in the cell extracts. Four conserved residues in the C-terminal loop of DPP8 (Phe(822), Val(833), Tyr(844), and His(859)), corresponding to those located at the dimer interface of DPP-IV, were individually mutated to Ala. Surprisingly, unlike DPP-IV, these single-site mutations abolished the enzymatic activity of DPP8 without disrupting its quaternary structure, indicating that dimerization itself is not sufficient for the optimal enzymatic activity of DPP8. Moreover, these mutations not only decreased k(cat), as did the corresponding DPP-IV mutations, but also dramatically increased K(m). We further show that the K(m) effect is independent of the substrate assayed. Finally, we identified the distinctive and strict substrate selectivity of DPP8 for hydrophobic or basic residues at the P2 site, which is in sharp contrast to the much less discriminative substrate specificity of DPP-IV. Our study has identified the residues absolutely required for the optimal activity of DPP8 and its unique substrate specificity. This study extends the functional importance of the C-terminal loop to the whole family of prolyl dipeptidases.
authors.affiliation: Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhu Nan Town, Miaoli Co., Taiwan 350, Republic of China.
authors.forename: Hong-Jen
authors.initials: HJ
authors.lastname: Lee
authors.forename: Yuan-Shou
authors.initials: YS
authors.lastname: Chen
authors.forename: Chi-Yuan
authors.initials: CY
authors.lastname: Chou
authors.forename: Chia-Hui
authors.initials: CH
authors.lastname: Chien
authors.forename: Chun-Hung
authors.initials: CH
authors.lastname: Lin
authors.forename: Gu-Gang
authors.initials: GG
authors.lastname: Chang
authors.forename: Xin
authors.initials: X
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D019281:Dimerization; D004150:Dipeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D004150:Dipeptidases; C487997:DPP8 protein, human