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J. Biol. Chem., Vol. 281, Issue 50, 38653-38662, December 15, 2006

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

Hong-Jen Lee, Yuan-Shou Chen, Chi-Yuan Chou, Chia-Hui Chien, Chun-Hung Lin^¶, Gu-Gang Chang, and Xin Chen¹

From the Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhu Nan Town, Miaoli 350, Faculty of Life Science, National Yang-Ming University, Taipei 112, and ^¶Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan

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 (Phe⁸²², Val⁸³³, Tyr⁸⁴⁴, and His⁸⁵⁹), 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.

Received for publication, April 24, 2006 , and in revised form, October 5, 2006.

^* This work was supported financially by National Research Program for Genomic Medicine Grant NSC95-3112-B-400-010 (to X. C.), the National Science Council, Taiwan, and National Health Research Institutes Grant BPR-094-PP-15 (to X. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1S and Fig. 1S.

¹ To whom correspondence should be addressed: Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhu Nan Town, Miaoli County, Taiwan 350, Republic of China. Tel.: 886-37-246166 (ext. 35718); Fax: 886-37-586456; E-mail: xchen{at}nhri.org.tw.

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