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Thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) from a human leukemic cell line has been purified to homogeneity with one-step affinity column chromatography. The purified enzyme has a specific activity of 3.8 jtmo /min per mg of protein, which corresponds to a turnover number of 250. These are the highest values reported for a thymidylate synthetase from neoplastic tissue. A ratio of 1.7 mol of 5-fluoro-2'.deoxyuridylate binds per mol of enzyme in the presence of 5,10-methylenetetrahydrofolate. The ternary complex so formed migrates intact on denaturing gels and can be precipitated with trichloroacetic acid; however, urea dissociates the ternary complex. The human thymidylate synthetase is composed of two subunits of 33,000 daltons each. It contains more residues of cysteine, glycine, and arginine and fewer of histidine than the well-studied thymidylate synthetase from Lactobacillus casei. Thymidylate synthetase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) has been the object of considerable interest because of its key function in mammalian DNA synthesis as the only de novo source of thymidylate. This enzyme is believed to be the major site of action of the cancer chemotherapeutic drug 5-fluorouracil due to inhibition by the metabolite 5-fluoro-2'-deoxyuridylate (FdUMP) (1). FdUMP, 5,10-methylenetetrahydrofolate (5,10-CH2H4PteGlu), and thymidylate synthetase form a ternary complex (2), which is joined by covalent bonds in some cases-i.e., is stable to protein denaturants and limited proteolysis (3)(4)(5)(6). In other cases, the ternary complex is dissociated partially (7) (16) and was used without purification. H4MTX was lyophilized in ampules, sealed in N2, and stored in the dark at -25°C.Aminoethyl polyacrylamide gel beads (Aminoethyl Bio-Gel P-150) were purchased from Bio-Rad, as were the reagents for polyacrylamide gel electrophoresis. Sephadex G-100 was obtained from Pharmacia. Other biochemicals were from Sigma.Cell Culture. CCRF-CEM human lymphoblastic leukemia cells (17) were thawed from mycoplasma-free stocks at 21/2-month intervals and were monitored routinely for absence of mycoplasma. These cells were grown in suspension in RPMI 1640 medium (GIBCO) supplemented with 10% fetal calf serum (GIBCO) and were harvested in late logarithmic growth, washed once with phosphate-buffered saline, and used immediately or stored in liquid N2.Affinity Column Preparation. Aminoethyl polyacrylamide (0.5 g) was hydrated with water and reacted with 20 mg of H4MTX and 150 mg of 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide-HCI by the method of Slavik et al. (12), except that N2 gas was used. About 40% of the resultant gel was transferred into a small plastic column (5-ml Pipetman tips partially stuffed with glass wool) in a cold room (40C). The light-tan beads were washed overnight with 200 ml of 2.5% NaHCO3/0.5 M KCl containing 0.15 M 2-mercaptoethanol. The columns were then washed with 100 ml of 10 mM potassium phosphate buffer (pH 7.4) c...

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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.