Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (12)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Kawase, S.
Right arrow Articles by Santi, D. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kawase, S.
Right arrow Articles by Santi, D. V.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Protein Engineering, Vol. 13, No. 8, 557-563, August 2000
© 2000 Oxford University Press

Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase

Shido Kawase, Sung-Woo Cho1, James Rozelle, Robert M. Stroud, Janet Finer-Moore and Daniel V. Santi2

Department of Biochemistry and Biophysics and Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94143-0448, USA and 1 Department of Biochemistry, College of Medicine, University of Ulsan, Seoul 138-040, Korea

Arginines R23, R178, R179 and R218 in thymidylate synthase (TS, EC 2.1.1.45) are hydrogen bond donors to the phosphate moiety of the substrate, dUMP. In order to investigate how these arginines contribute to enzyme function, we prepared complete replacement sets of mutants at each of the four sites in Lactobacillus casei TS. Mutations of R23 increase Km for dUMP 2–20-fold, increase Km for cofactor 8–40-fold and decrease kcat 9–20-fold, reflecting the direct role of the R23 side chain in binding and orienting the cofactor in ternary complexes of the enzyme. Mutations of R178 increase Km for dUMP 40–2000-fold, increase Km for cofactor 3–20-fold and do not significantly affect kcat. These results are consistent with the fact that this residue is an integral part of the dUMP-binding wall and contributes to the orientation and ordering of several other dUMP binding residues. Kinetic parameters for all R179 mutations except R179P were not significantly different from wild-type values, reflecting the fact that this external arginine does not directly contact the cofactor or other ligand-binding residues. R218 is essential for the structure of the catalytic site and all mutations of this arginine except R218K were inactive.


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


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
D. Hamelberg, T. Shen, and J. A. McCammon
A proposed signaling motif for nuclear import in mRNA processing via the formation of arginine claw
PNAS, September 18, 2007; 104(38): 14947 - 14951.
[Abstract] [Full Text] [PDF]

Home page
 Protein Eng Des SelHome page
D. Gonzalez-Pacanowska, L. M. Ruiz-Perez, M. A. Carreras-Gomez, M. P. Costi, R. M. Stroud, J. Finer-Moore, and D. V. Santi
The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase
Protein Eng. Des. Sel., August 1, 2003; 16(8): 607 - 614.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.