Protein Engineering, Vol. 16, No. 3, 229-240,
March 2003
© 2003 Oxford University Press
The only active mutant of thymidylate synthase D169, a residue far from the site of methyl transfer, demonstrates the exquisite nature of enzyme specificity
Department of Biochemistry and Biophysics and Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA 94143-0448, USA
1 To whom correspondence should be addressed. Present address: S412C UCSF-GENENTECH Hall, 600 16th Street, San Francisco, CA 94143-2240, USA
Cysteine is the only variant of D169, a cofactor-binding residue in thymidylate synthase, that shows in vivo activity. The 2.4 Å crystal structure of Escherichia coli thymidylate synthase D169C in a complex with dUMP and the antifolate CB3717 shows it to be an asymmetric dimer, with only one active site covalently bonded to dUMP. At the active site with covalently bound substrate, C169 S
adopts the roles of both carboxyl oxygens of D169, making a 3.6 Å S•••H–N hydrogen bond to 3-NH of CB3717 and a 3.4 Å water-mediated hydrogen bond to H212. Analogous hydrogen bonds formed during the enzyme reaction are important for cofactor binding and are postulated to contribute to catalysis. The C169 side chain is likely to be ionized, making it a better hydrogen bond acceptor than a neutral sulfhydryl group. At the second active site, C169 S makes a shorter (3 Å) hydrogen bond to the 3-NH of CB3717, CB3717 is ~1.5 Å out of its binding site and there is no covalent bond between dUMP and the catalytic cysteine. Changes to partitioning among productive and non-productive conformations of reaction intermediates may contribute as much, if not more, to the diminished activity of this mutant than reduced stabilization of transition states.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Shao, X. Xu, N. Jing, and D. J. Tweardy Unique structural determinants for stat3 recruitment and activation by the granulocyte colony-stimulating factor receptor at phosphotyrosine ligands 704 and 744. J. Immunol., March 1, 2006; 176(5): 2933 - 2941. [Abstract] [Full Text] [PDF]
|
|