Heterodimeric thymidylate synthases with C-terminal deletion on one subunit

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Thymidylate synthase (TS) is a critical enzyme in DNA synthesis.
• The enzyme functions as a dimer, with residues from one subunit often essential for the active site of the opposing subunit.
• Previous studies indicated that removing the C-terminal valine from either or both subunits inactivates the enzyme.

Purpose of the Study:

• To investigate the activity of heterodimeric thymidylate synthases where only one subunit lacks the C-terminal valine.
• To explore the role of the C-terminal valine and the opposing active site residue (Arg-178) in thymidylate synthase function.

Main Methods:

• Site-directed mutagenesis was used to create specific TS mutants (V316Am and R178F).
• Reversible unfolding and subunit reassociation techniques were employed to form heterodimeric TS.
• Enzyme kinetics (kcat, Km) were measured for wild-type and heterodimeric enzymes.

Main Results:

• Heterodimeric TS with V316Am in only one subunit (V316Am-WT) exhibited approximately half the activity of wild-type TS.
• The R178F-V316Am heterodimer, containing one intact active site, also showed half the wild-type activity.
• Both heterodimeric enzymes displayed Km values similar to wild-type TS, indicating comparable substrate and cofactor binding.

Conclusions:

• The C-terminal valine is not strictly required in both subunits for thymidylate synthase activity.
• Heterodimeric TS with a single C-terminal valine can be catalytically active.
• These findings provide insights into the structural and functional requirements of the TS dimer.