Differences in natural ligand and fluoropyrimidine binding to human thymidylate synthase identified by transient-state spectroscopic and continuous variation methods.
Thymidylate synthase (TS) is a central target for the design of chemotherapeutic agents due to its vital role in DNA synthesis. Structural studies of binary complexes between Escherichia coli TS and various nucleotides suggest the chemotherapeutic agent FdUMP and the natural ligand dUMP bind similarly. We show, however, that FdUMP binding to human TS yields a substantially greater decrease in fluorescence than does dUMP. Because the difference in quenching due to ligand binding was approximately two-fold and this difference was not seen when using ecTS, the intriguing result indicated a significant difference in the mode of FdUMP binding to the human enzyme. We compared the binding affinities of dUMP, FdUMP, and TMP to TS from both species and found no significant differences for the individual ligands. Because binding affinities were not different among the ligands, the method of continuous variation was employed to determine binding stoichiometry. Similar to that found for dUMP binding to human and ecTS, FdUMP displayed single site occupancy with both enzymes. These results show that nucleotide binding differences exist for FdUMP and dUMP binding to the human enzyme. The observed differences are not due to differences in stoichiometry or ligand affinity. Therefore, although the crystal structure of human TS with various nucleotide ligands has not been solved, these results show that the differences observed using fluorescence methods result from as yet unidentified differential interactions between the human enzyme and nucleotide ligands.
Felder
Takita
T
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, USA.
Dunlap
R Bruce
RB
Dix
Daniel
D
Spencer
Trent
T
D001665:Binding Sites; D003856:Deoxyuracil Nucleotides / Q000737:chemistry; D005453:Fluorescence; D005468:Fluorodeoxyuridylate / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D011743:Pyrimidines / Q000737:chemistry*; D013053:Spectrophotometry / Q000379:methods; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000737:chemistry*
D003856:Deoxyuracil Nucleotides; D008024:Ligands; D011743:Pyrimidines; D005468:Fluorodeoxyuridylate; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase