Thymidylate synthase (TS) catalyzes the conversion of dUMP and [6R]-5,10- methylenetetrahydrofolate (CH2H4folate) to dTMP and H2folate by reductive methylation, in which CH2H4folate is both methyl donor and reducing agent. This reaction provides the only de novo source of DTMP which is required for DNA synthesis. Thus maintaining adequate levels of TS activity is usually critical in rapidly growing cells where DNA synthesis is correspondingly rapid. This enzyme is therefore an important target for chemotherapeutic agents such as the anti-tumor agent 5-fluoro-2'- deoxyuridylate (FdUMP) so that TS is of interest both as a model for enzymatic catalysis and for its clinical relevance. The broad objective of this proposal is to elucidate at the molecular level the relationship between the structure and function of thymidylate synthase (TS) from both bacterial (Escherichia coli) and human sources with respect to catalysis, binding of inhibitors, and interaction with other intracellular enzymes.
The specific aims are to (1) describe the detailed kinetic mechanism for catalysis by TS in which values of equilibrium and rate constants for all steps in the catalytic cycle will be determined with either CH2H4folate or [6R]-5,10-CH2-H4pteroyl-gamma-polyglutamates (the natural intracellular form) as folate substrate; (2) correctly determine the kinetics and thermodynamics of FdUMP binding to TS in binary (TS.FdUMP), ternary (TS.FdUMP.CH2H4folate), and covalent ternary (TS-FdUMP- CH2H4folate) complexes, (3) evaluate the role of specific amino acid side chairs in catalysis and in binding of FdUMP, and (4) determine the role of specific interactions between Ts and dihydrofolate reductase (DHFR) in catalysis and inhibition. Rate and equilibrium constants for individual steps in the catalytic cycle and in formation of complexes with FdUMP will be measured employing appropriate techniques including stopped-flow spectroscopy and quenched- flow methods. These constants will subsequently be used in construction of kinetic models for catalysis that will be tested by their ability to predict values of steady-state catalytic parameters. Hypotheses concerning the roles of specific amino acid side chains will be tested by comparing individual steps in catalysis and formation of inhibitory complexes for altered enzymes generated by oligonucleotide-directed mutagenesis in which single amino acid substitutions have been made with comparable steps in wild-type enzyme. Interactions between TS and DHFR will be evaluated by comparing catalysis by each enzyme alone with that when both enzymes are present and by determining the affinity of these enzymes for one another.
| Spencer, H T; Villafranca, J E; Appleman, J R (1997) Kinetic scheme for thymidylate synthase from Escherichia coli: determination from measurements of ligand binding, primary and secondary isotope effects, and pre-steady-state catalysis. Biochemistry 36:4212-22 |
