Interaction of 5-fluoro-4-thio-2'-deoxyuridine 5'-phosphate with mammalian tumour thymidylate synthase: role of the pyrimidine N(3)-H dissociation.
The role of the pyrimidine N(3)-H in binding of dUMP derivatives to thymidylate synthase was evaluated with the aid of a new dUMP analogue, 5-fluoro-4-thio-dUMP, synthesized by an improved thiation and enzymatic phosphorylation. The interaction of this analogue, and of 5-FdUMP, with the enzyme, and the pH-dependence of these interactions, were compared. Both were slow-binding competitive inhibitors of the enzyme from Ehrlich carcinoma, L1210 and CCRF-CEM cells, with Ki an order of magnitude higher for 5-fluoro-4-thio-dUMP than for 5-FdUMP. With both nucleotides, as well as the parent nucleosides, enzyme inactivation increased as the pH was lowered from 8 to 6. Maximum inactivation with 5-FdUrd was at pH 7.0, and with 5-fluoro-4-thio-dUrd at pH 6.0, in agreement with the higher pKa for the N(3)-H dissociation of the former, and pointing to participation of the N(3)-H as a hydrogen donor in binding to the enzyme.
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology; D003856:Deoxyuracil Nucleotides / Q000494:pharmacology*; D005468:Fluorodeoxyuridylate / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007939:Leukemia L1210 / Q000201:enzymology; D007945:Leukemia, Lymphoid / Q000201:enzymology / Q000473:pathology; D051379:Mice; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013873:Thionucleotides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism
D003856:Deoxyuracil Nucleotides; D009363:Neoplasm Proteins; D013873:Thionucleotides; C054543:5-fluoro-4-thio-2'-deoxyuridylate; D005468:Fluorodeoxyuridylate; D013940:Thymidylate Synthase