Irreversible inhibition of thymidylate synthase by pyridoxine (B6) analogues.

Three vitamin B6 analogues have been synthesized and tested as inhibitors of thymidylate synthase. The compounds are: 4',5'-dichloro-, 4',5'-dibromo- and 4',5'-diiodo-pyridoxine. All three analogues inhibited the enzyme irreversibly. The kinetic data for the chloro- and bromo-analogues showed that a limiting rate of inhibition is approached as the inhibitor concentration is increased, which indicates that a reversible enzyme:inhibitor affinity complex is formed prior to the irreversible reaction. 4',5'-Dibromo-pyridoxine exhibited a greater binding affinity (lower Ki) for thymidylate synthase than 4',5'-dichloro-pyridoxine, and it also reacted faster to irreversibly inhibit the enzyme. The presence of the substrate dUMP (10 microM) completely protected thymidylate synthase from inhibition. These data suggest that the halogenated vitamin B6 analogues are active site-directed inhibitors of thymidylate synthase, which first bind reversibly to the catalytic site and then react irreversibly with the enzyme.