Thymidylate synthases biosynthesize DNA and catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate to form 2'-deoxythymidine-5'-monophosphate. Humans rely on classical thymidylate synthases, whereas many human pathogens produce a novel type of Flavin-dependent thymidylate synthases (FDTSs). Although several studies have investigated FDTS catalysis, the kinetic mechanism, is still currently under investigation. One of the current problems of constructing a complete kinetic model is the amount of reaction conditions and time required for analyzing each reaction. In our research study, a tritium release assay is being developed to analyze multiple reaction conditions simultaneously, providing an efficient way to a construct multi-substrate kinetic model.  Compared product formation indicated by release of tritium from 5-3H-dUMP into the water to the accepted 14C-dUMP assay will validate this assay for following FDTS activity.  Kinetic studies will be important in resolving the binding and release order of substrates and products, which in turn will aid in rational drug development.

Developing the tritium release assay for kinetics measurement of flavin-dependent thymidylate synthase

243rd National Meeting (2012)

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