Partitioning roles of side chains in affinity, orientation, and catalysis with structures for mutant complexes: Asparagine-229 in thymidylate synthase

Janet S. Finer-Moore, Lu Liu, Christian E. Schafmeister, David L. Birdsall, Ted Mau, Daniel V. Santi, Robert M. Stroud

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Abstract

Thymidylate synthase (TS) methylates only dUMP, not dCMP. The crystal structure of TS·dCMP shows dCMP 4-NH2 excluded from the space between Asn- 229 and His-199 by the hydrogen bonding and steric properties of Asn-229. Consequently, 6-C of dCMP is over 4 A from the active site sulfhydryl. The Asn-229 side chain is prevented from flipping 180° to an orientation that could hydrogen bond to dCMP by a hydrogen bond network between conserved residues. Thus, the specific binding of dUMP by TS results from occlusion of competing substrates by steric and electronic effects of residues in the active site cavity. When Ash-229 is replaced by a cysteine, the Cys-229 Sγ rotates out of the active site, and the mutant enzyme binds both dCMP and dUMP tightly but does not methylate dCMP. Thus simply admitting dCMP into the dUMP binding site of TS is not sufficient for methylation of dCMP. Structures of nucleotide complexes of TS N229D provide a reasonable explanation for the preferential methylation of dCMP instead of dUMP by this mutant. In TS N229D·dCMP, Asp-229 forms hydrogen bonds to 3-N and 4-NH2 of dCMP. Neither the Asp-229 carboxyl moiety nor ordered water appears to hydrogen bond to 4- O of dUMP. Hydrogen bonds to 4-O (or 4-NH2) have been proposed to stabilize reaction intermediates. If their absence in TS N229D°dUMP persists in the ternary complex, it could explain the 104-d decrease in k(cat)/K(m) for dUMP.

Original languageEnglish (US)
Pages (from-to)5125-5136
Number of pages12
JournalBiochemistry
Volume35
Issue number16
DOIs
Publication statusPublished - Apr 23 1996

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ASJC Scopus subject areas

  • Biochemistry

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