Use of strain in a stereospecific catalytic mechanism: Crystal structures of Escherichia coli thymidylate synthase bound to FdUMP and methylenetetrahydrofolate

David C. Hyatt, Frank Maley, William "Bill" Montfort

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Abstract

Two crystal structures for E. coli thymidylate synthase (TS) bound to the mechanism-based inhibitor 5-fluoro-dUMP (FdUMP) and methylenetetrahydrofolate (CH2THF) have been determined to 2.6 and 2.2 Å nominal resolutions, with crystallographic R factors of 0.180 and 0.178, respectively. The inhibitor and cofactor are well ordered in both structures and display covalent links to each other and to Cys 146 in the TS active site. The structures are in general agreement with a previous report for this complex (D. A. Matthews et al. (1990) J. Mol. Biol. 214, 937-948), but differ in two key respects: (i) the methylene bridge linking FdUMP and CH2THF is rotated about 60°to a different position and (ii) the electron density for C6 of FdUMP, which is covalently linked to Cys 146, is more diffuse than for the other atoms in the pyrimidine ring. The ligand arrangement observed in the previous structure led the authors to propose that a large conformational change in ligand geometry must occur in order to facilitate catalysis and yield the correct chirality in the methyl of product dTMP. The new structures suggest a different mechanism for product formation that does not require ligands to greatly alter their conformations during catalysis and which makes use of instability in the nucleotide-Cys 146 thiol adduct to avoid a deep free energy well and assist in proton abstraction from dUMP. All intermediates in the proposed mechanism were modeled and energy minimized in the TS active site, and all can be accommodated in the present structures. The role of ligand-induced conformational change in the TS mechanism and the possibility of Tyr 94 acting as a base during catalysis are also discussed.

Original languageEnglish (US)
Pages (from-to)4585-4594
Number of pages10
JournalBiochemistry
Volume36
Issue number15
DOIs
StatePublished - Apr 15 1997

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Thymidylate Synthase
Escherichia coli
Crystal structure
Catalysis
Ligands
Catalytic Domain
R Factors
Chirality
Sulfhydryl Compounds
Free energy
Carrier concentration
Conformations
Protons
Nucleotides
Electrons
Atoms
Geometry
2'-deoxyuridylic acid

ASJC Scopus subject areas

  • Biochemistry

Cite this

Use of strain in a stereospecific catalytic mechanism : Crystal structures of Escherichia coli thymidylate synthase bound to FdUMP and methylenetetrahydrofolate. / Hyatt, David C.; Maley, Frank; Montfort, William "Bill".

In: Biochemistry, Vol. 36, No. 15, 15.04.1997, p. 4585-4594.

Research output: Contribution to journalArticle

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