Formation of ternary complexes of thymidylate synthetase as followed by absorbance, fluorescence, and circular dichroic spectra and gel electrophoresis.

Ternary complexes of thymidylate synthetase (Form II and Form III), which are composed of the enzyme, 5-fluorodeoxyuridylate, and the natural isomer of methylenetetrahydrofolate, were generated by titrating thymidylate synthetase (Form I) in the presence of 5-fluorodeoxyuridylate with either the enzymatically prepared natural isomer or the chemically prepared racemic mixture of the diastereomers of methylenetetrahydrofolate. Such titrations were monitored by absorption, circular dichroic and fluorescence spectroscopy, and polyacrylamide gel electrophoresis. The results of these investigations suggest that the natural isomer of methylenetetrahydrofolate is primarily involved in the formation of stable ternary complexes with thymidylate synthetase and 5-fluorodeoxyuridylate but that the unnatural isomer of methylenetetrahydrofolate, when present in solution, may compete with the natural isomer by forming relatively weak complexes with the enzyme and the 5-fluorodeoxyuridylate.