Interaction of pyridoxal phosphate with thymidylate synthase: spectral and equilibrium dialysis studies

Abstract

1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents. 2. The dissociation constant of the thymidylate synthase-PLP complex determined by equilibrium dialysis was 9 +/- 1.6 microM, the maximum number of PLP molecules bound per molecule of native thymidylate synthase was 2.5 +/- 0.4, and the Hill coefficient was 0.97. 3. No evidence of PLP binding was found with denatured thymidylate synthase, and only slight binding was observed when enzyme SH groups were blocked or when the active site was blocked with 5-fluorodeoxyuridylate (FdUMP) and methylene tetrahydrofolate. 4. The presence of dUMP, dTMP, or FdUMP interfered with the binding of PLP to thymidylate synthase, and the presence of equimolar amounts of PLP interfered with the binding of dUMP.