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Positive Cooperativity in Substrate Binding by Human Thymidylate Synthase.

Jeffrey P Bonin1, Paul J Sapienza2, Emily Wilkerson3

  • 1Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Biophysical Journal
|September 11, 2019
PubMed
Summary

Human thymidylate synthase (TS) exhibits positive cooperativity in deoxyuridine monophosphate (dUMP) binding, challenging previous findings. This discovery impacts understanding of DNA replication and cancer drug targets.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Thymidylate synthase (TS) is crucial for DNA replication, producing dTMP from dUMP.
  • TS is a homodimer and a target for cancer therapeutics.
  • Allosteric communication between TS active sites suggests cooperativity in substrate binding.

Purpose of the Study:

  • To investigate the binding cooperativity of deoxyuridine monophosphate (dUMP) with human thymidylate synthase (TS).
  • To clarify conflicting reports on negative cooperativity in TS.
  • To provide thermodynamic characterization of dUMP binding to human TS.

Main Methods:

  • Isothermal titration calorimetry (ITC) to measure binding thermodynamics.
  • Two-dimensional (2D) lineshape analysis of NMR titration spectra.
  • Global fitting of isotherms and accurate determination of protein concentration for calorimetric analysis.

Main Results:

  • Human TS binds dUMP with approximately 9-fold entropically driven positive cooperativity (ρ_ITC = 9 ± 1, ρ_NMR = 7 ± 1).
  • This finding contrasts with previously reported negative cooperativity in other species.
  • Initial isotherm curvature in calorimetry can indicate positive cooperativity; 2D NMR is valuable for quantifying binding cooperativity.

Conclusions:

  • Human TS displays positive cooperativity in dUMP binding, driven by entropy.
  • Accurate thermodynamic characterization requires careful experimental design and data analysis, including global fitting.
  • 2D NMR is a powerful tool for assessing binding cooperativity, especially with unique bound intermediate resonances.