Bacterial Thymidylate Synthase Binds Two Molecules of Substrate and Cofactor without Cooperativity

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Thymidylate synthase (TSase) is a critical enzyme for DNA synthesis, making it a key drug target.
• TSase exhibits
• half-the-sites reactivity,
• a form of negative cooperativity with an unclear mechanistic basis.

Purpose of the Study:

• To thermodynamically dissect the multisite binding of dUMP to E. coli TSase.
• To resolve conflicting reports on the basis of TSase's half-the-sites reactivity.

Main Methods:

• Isothermal Titration Calorimetry (ITC) to analyze binding thermodynamics.
• Nuclear Magnetic Resonance (NMR) titration to study subunit interactions and complex formation.

Main Results:

• dUMP binds to free and singly bound E. coli TSase with nearly equal affinity.
• Thermodynamic analysis reveals minimal allosteric effects (ΔG°bind) between binding sites.
• NMR data indicate minor intersubunit cooperativity in ternary complex formation with 5F-dUMP and cofactor.

Conclusions:

• Functional communication between TSase subunits is minimal during both substrate binding and ternary complex formation.
• The observed half-the-sites reactivity is not primarily driven by significant allosteric effects in binding or initial complex formation.