Interaction of Thymidylate Synthase with Pyridoxal 5′-Phosphate As Studied by UV/Visible Difference Spectroscopy and Molecular Modeling

Daniel V. Santi, Tian Mei Ouyang, Anthony K. Tan, Donald H. Gregory, Thomas Scanlan, Christopher W. Carreras

Research output: Contribution to journalArticle

22 Scopus citations

Abstract

Pyridoxal 5′-phosphate (PLP) is an effective inhibitor of Lactobacillus casei thymidylate synthase (TS), competitive with respect to the nucleotide substrate dUMP (Chen et al., 1989). The UV/vis difference spectra of TS-PLP complexes show λmax at 328 nm due to the specific interaction between Cys 198 of TS and PLP to form a thiohemiacetal, and λmin at 388 nm due to depletion of free PLP. At high concentrations of PLP a new absorbance at 430 nm forms due to nonspecific Schiff base formation between PLP and lysine residues of the enzyme. Using spectral titration at 328 nm, the binding constant of the specific TS-PLP complex was determined to be 0.5 µM, and the stoichiometry was 2 mol of PLP/mol of TS dimer. The 328-nm absorbance of the TS-PLP complex can be competitively and completely eliminated by addition of dUMP or dTMP; this serves as a convenient binding assay for molecules which bind to the active site of TS. Analogs of PLP which do not contain the phosphate or the aldehyde moieties of PLP bound poorly to the enzyme, thus demonstrating the importance of these functional groups for binding. When treated with PLP, C244T TS, which contains the active site Cys 198 as the sole cysteine residue, showed the same properties as the wild-type enzyme. Treatment of the C198 A and C198S mutants with PLP did not produce the absorbance at 328 nm assigned to thiohemiacetal formation. Molecular modeling studies showed that when the phosphate of PLP is docked into the phosphate binding site of TS, the aldehyde carbon of PLP is perfectly positioned to form a thiohemiacetal with the catalytic thiol of Cys 198. Side chains of amino acid residues of the protein which are close to or may interact with PLP were tentatively assigned.

Original languageEnglish (US)
Pages (from-to)11819-11824
Number of pages6
JournalBiochemistry
Volume32
Issue number44
DOIs
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry

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