Protein Science Sheba protein
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Protein Science (2008), 17:240-250. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 The Protein Society
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Metzler, W. J.
Right arrow Articles by Marcinkeviciene, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Metzler, W. J.
Right arrow Articles by Marcinkeviciene, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Involvement of DPP-IV catalytic residues in enzyme–saxagliptin complex formation

William J. Metzler1, Joseph Yanchunas1, Carolyn Weigelt1, Kevin Kish1, Herbert E. Klei1, Dianlin Xie1, Yaqun Zhang1, Martin Corbett1, James K. Tamura1, Bin He2, Lawrence G. Hamann3, Mark S. Kirby2, and Jovita Marcinkeviciene1

1 Department of Molecular Biosciences, Bristol-Myers Squibb Research and Development, Princeton, New Jersey 08543-4000, USA
2 Department of Metabolic Diseases, Bristol-Myers Squibb Research and Development, Princeton, New Jersey 08543-4000, USA
3 Department of Discovery Chemistry, Bristol-Myers Squibb Research and Development, Princeton, New Jersey 08543-4000, USA

(RECEIVED September 18, 2007; FINAL REVISION November 8, 2007; ACCEPTED November 8, 2007)

The inhibition of DPP-IV by saxagliptin has been proposed to occur through formation of a covalent but reversible complex. To evaluate further the mechanism of inhibition, we determined the X-ray crystal structure of the DPP-IV:saxagliptin complex. This structure reveals covalent attachment between S630 and the inhibitor nitrile carbon (C–O distance <1.3 Å). To investigate whether this serine addition is assisted by the catalytic His-Asp dyad, we generated two mutants of DPP-IV, S630A and H740Q, and assayed them for ability to bind inhibitor. DPP-IVH740Q bound saxagliptin with an ~1000-fold reduction in affinity relative to DPP-IVWT, while DPP-IVS630A showed no evidence for binding inhibitor. An analog of saxagliptin lacking the nitrile group showed unchanged binding properties to the both mutant proteins, highlighting the essential role S630 and H740 play in covalent bond formation between S630 and saxagliptin. Further supporting mechanism-based inhibition by saxagliptin, NMR spectra of enzyme–saxagliptin complexes revealed the presence of three downfield resonances with low fractionation factors characteristic of short and strong hydrogen bonds (SSHB). Comparison of the NMR spectra of various wild-type and mutant DPP-IV:ligand complexes enabled assignment of a resonance at ~14 ppm to H740. Two additional DPP-IV mutants, Y547F and Y547Q, generated to probe potential stabilization of the enzyme–inhibitor complex by this residue, did not show any differences in inhibitor binding either by ITC or NMR. Together with the previously published enzymatic data, the structural and binding data presented here strongly support a histidine-assisted covalent bond formation between S630 hydroxyl oxygen and the nitrile group of saxagliptin.

Keywords: DPP-IV; X-ray crystal structure; mutant; ITC; proton NMR; short, strong hydrogen bond; serine protease; saxagliptin


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?




HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2008 by The Protein Society.