Hydrodynamic behavior of human and bacterial thymidylate synthetases and thymidylate synthetase--5-fluoro-2'-deoxyuridylate--5,10-methylenetetrahydrofolate complexes. Evidence for large conformational changes during catalysis.
The conformations of thymidylate synthetases from CCRF-CEM human leukemic cells and Lactobacillus casei were studied by hydrodynamic methods. Although the human enzyme has a molecular weight of 70 000--72 500, somewhat smaller than that of the L. casei enzyme, it has a larger Stokes radius and a lower sedimentation coefficient, indicating that the human enzyme is less spherical than the bacterial enzyme. Thymidylate synthetases from the human leukemic cells and the bacterial source both undergo substantial conformational changes upon the formation of a covalent ternary complex with the mechanism-based inhibitor 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate. The Stokes radius of both proteins decreases by 3.5% when the ternary complex is formed in spite of the 1.8% increase in molecular weight, and the sedimentation coefficient increases by 3.5% after appropriate corrections for the bound ligands. Ternary complex formation results in a more compact structure for both enzymes, with approximately the same reduction in the frictional ratio. Experiments with the bacterial enzyme indicate that approximately 70% of the total conformational change occurs upon binding of 1 mol of ligands/mol of enzyme. These results demonstrate that human and bacterial thymidylate synthetases undergo marked structural changes upon forming a ternary complex which is probably very similar to an activated complex formed with both substrates. These investigations also provide evidence for fundamental similarities in the mechanism of ternary complex formation with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate, despite the marked differences in amino acid composition and the dissimilar conformations of these two enzymes obtained from widely divergent sources.
D001665:Binding Sites; D002460:Cell Line; D003856:Deoxyuracil Nucleotides / Q000494:pharmacology*; D005468:Fluorodeoxyuridylate / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D007945:Leukemia, Lymphoid; D008780:Methyltransferases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013045:Species Specificity; D013763:Tetrahydrofolates / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000302:isolation & purification / Q000378:metabolism*
D003856:Deoxyuracil Nucleotides; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D005468:Fluorodeoxyuridylate; D008780:Methyltransferases; D013940:Thymidylate Synthase