Investigation of the Dimer Interface and Substrate Specificity of Prolyl Dipeptidase DPP8

Xin Chen,Yuan-Shou Chen,Chi-Yuan Chou,Chun-Hung Lin,Chia-Hui Chien,Hong-Jen Lee,Gu-Gang Chang

doi:10.1074/jbc.m603895200

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.

Highlights

Kines, and neuropeptides contain one or more proline residues
For substrate scanning experiments, purified DPP8 (80 nM) or dipeptidyl peptidase IV (DPP-IV) (20 nM) proteins were incubated in phosphate-buffered saline with 10 ␮M individual chromogenic substrate X-Pro-pNA or Ala-X-pNA, where X is any of the 20 amino acids
No differences were observed in the biochemical properties, including the quaternary structures and enzymatic activity, of this version of DPP8 and that previously described with only a His tag [28]

Summary

EXPERIMENTAL PROCEDURES

Materials—The chromogenic dipeptide substrates were purchased from Bachem. X-Pro-pNA and Ala-X-pNA libraries, in which X is any amino acid, were custom synthesized by GL Biochem Ltd. (Shanghai, China). The BamHI-NotI fragment of pTOPO-MHFDPP8 was further subcloned into the BamHI and NotI sites of pBAC-PAK8 [28], generating plasmid pBAC-MHF-DPP8 for the expression of DPP8 protein in baculovirus-infected insect cells. The proteins were transferred to polyvinylidene difluoride membrane, and Western blot analysis was performed as described previously, with anti-DPP8 antibody, which was generated in house by immunizing a rabbit with purified DPP8 protein from baculovirus-infected insect cells [28]. Determination of Substrate Specificities and Kinetic Constants of DPP8s—DPP-IV protein was purified as described previously [13]. For substrate scanning experiments, purified DPP8 (80 nM) or DPP-IV (20 nM) proteins were incubated in phosphate-buffered saline with 10 ␮M individual chromogenic substrate X-Pro-pNA or Ala-X-pNA, where X is any of the 20 amino acids.

RESULTS

30 Ϯ 6 kcat sϪ1

DISCUSSION

72 Ϯ 4 102 Ϯ 9