Relative affinity of 5,10-methylenetetrahydrofolylpolyglutamates for the Lactobacillus casei thymidylate synthetase-5-fluorodeoxyuridylate binary complex

The affinity of a series of methylenetetrahydrofolate polyglutamates for the binary complex of Lactobacillus casei thymidylate synthetase and fluorodeoxyuridylate has been investigated by kinetic and equilibrium techniques. The relative rates of binding for polyglutamates with one through seven glutamate residues were determined at 0 °C. Reactions were stopped by quenching into sodium dodecyl sulfate with subsequent determination of bound tritiated fluorodeoxyuridylate by gel filtration chromatography. Rates increased up to five residues beyond which a slight decrease occurred. Relative equilibrium binding affinities for all possible pairs of polyglutamates from the same series were determined by electrophoretic separation of tritiated complexes. In every case, the longer chain length member of the pair was bound more tightly. Complexes involving only a single subunit of thymidylate synthetase were compared with those in which both subunits were bound. Monomeric (1:1:1) complexes invariably showed a greater affinity for the longer chain length member of the pair than the corresponding dimeric (2:2:1) species. These results are interpreted in terms of possible binding site models for thymidylate synthetase.