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Thymidylate synthase with a C-terminal deletion catalyzes partial reactions but is unable to catalyze thymidylate

C W Carreras1, S C Climie, D V Santi

  • 1Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0448.

Biochemistry
|July 7, 1992
PubMed

Insights

The Lactobacillus casei thymidylate synthase V316Am mutant, lacking a C-terminal valine, cannot form dTMP but retains partial catalytic activity. This indicates the mutation affects later steps in the reaction mechanism, not initial cofactor binding.

Area of Science:

  • Enzymology
  • Biochemistry
  • Molecular Biology

Background:

  • Lactobacillus casei thymidylate synthase (TS) is crucial for DNA synthesis.
  • Mutations can reveal insights into enzyme mechanisms and active site residues.

Purpose of the Study:

  • To investigate the catalytic mechanism of the V316Am mutant of Lactobacillus casei TS.
  • To determine the role of the C-terminal valine in TS catalysis and substrate binding.

Main Methods:

  • Enzyme kinetics assays were performed on the V316Am mutant.
  • Catalytic activity, inhibition patterns, and complex formation were analyzed.
  • Comparison with wild-type TS was conducted.

Main Results:

  • V316Am lacks dTMP formation activity but retains partial reactions: 5-hydrogen exchange and dehalogenation.
  • Similar kcat and Km values for partial reactions suggest intact substrate binding and covalent catalysis.
  • Competitive inhibition by dUMP, dTMP, and FdUMP indicates preserved nucleotide binding.
  • V316Am forms a covalent ternary complex, but with reduced cofactor affinity and stability.

Conclusions:

  • The C-terminal valine is not essential for substrate binding or initial covalent bond formation.
  • The V316Am mutation primarily affects steps subsequent to cofactor binding in the TS catalytic cycle.

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