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Related Experiment Videos

Subunit complementation of thymidylate synthase.

M Pookanjanatavip1, Y Yuthavong, P J Greene

  • 1Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.

Biochemistry
|October 27, 1992
PubMed
Summary
This summary is machine-generated.

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Inactive thymidylate synthase mutants were combined to create functional enzyme hybrids. This subunit complementation approach successfully restored enzyme activity in both in vivo and in vitro experiments, demonstrating a novel method for enzyme engineering.

Area of Science:

  • Biochemistry
  • Enzyme kinetics
  • Protein engineering

Background:

  • Thymidylate synthase is a crucial enzyme in DNA synthesis.
  • Its active sites are formed by residues from both subunits, a phenomenon known as subunit complementation.
  • Understanding this interaction is key to developing new therapeutic strategies.

Purpose of the Study:

  • To test the hypothesis that inactive thymidylate synthase mutants can form active heterodimeric hybrids.
  • To investigate the feasibility of in vivo and in vitro complementation of thymidylate synthase subunits.
  • To characterize the catalytic efficiency of the reconstituted enzyme.

Main Methods:

  • Co-transformation of plasmid mixtures encoding inactive Arg-178 and Cys-198 mutants in a thymidylate synthase-deficient host for in vivo complementation.

Related Experiment Videos

  • Selection and screening for transformants exhibiting growth in the absence of thymidine.
  • Reversible dissociation and unfolding of purified mutant proteins followed by refolding for in vitro complementation.
  • Determination of kinetic parameters (Km, kcat) for the heterodimeric enzyme.

    • Main Results:

    • Successful subunit complementation was observed in vivo and in vitro, generating active thymidylate synthase from inactive mutants.
    • A wide variety of Arg-178 and Cys-198 mutants supported complementation.
    • The R178F + C198A heterodimer exhibited kinetic parameters (Km) comparable to the wild-type enzyme.
    • The catalytic efficiency (kcat) of the heterodimer approached that of the wild-type enzyme.

    Conclusions:

    • Inactive subunits of thymidylate synthase can indeed complement each other to form a functional active site.
    • This subunit complementation strategy offers a viable method for engineering active enzymes from inactive components.
    • The findings provide insights into the cooperative nature of thymidylate synthase active sites and potential applications in enzyme engineering.