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

Abstract

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.

Ligand-Induced Conformational Changes of Thymidylate Synthase Detected by Limited Proteolysis

“…Danenberg and Danenberg (1979) have shown that FdUMP and CHrlLfolate binding to one catalytic site induced conformational changes that exposed the other catalytic site. Also, the formation of the ternary complex of thymidylate synthase with FdUMP and CH2-H4folate produced changes in the hydrodynamic properties of the enzyme, as measured by gel filtration and density gradient centrifugation, that were consistent with large conformational changes resulting in a more compact structure (Lockshin & Danenberg, 1980). The changes in absorption, fluorescence, and circular dichroic spectra that accompany ternary complex formation are consistent with conformational changes, though other interpretations are possible (Danenberg et al, 1974;Santi et al, 1974;Galivan et al, 1975;Donato et al, 1976; Manavalan et al, 1986).…”

Fluorinated Pyrimidines and Their Nucleosides

“…First, the difference in conformation between the free enzyme and the ternary complex can be easily observed by Sephadex gel filtration and high-speed centrifugation. The Stokes radius of TS from both human and bacterial sources was found to decrease by 3.5% (with a similar increase in sedimentation coefficient) upon ternary complex fromation, in spite of a 1.8% increase in molecular weight (161). Changes in conformation of this magnitude are normally associated with multisubunit allosterically controlled enzymes such as aspartate transcarbamylase (162).…”

Use of a purified heterodimer to test negative cooperativity as the basis of substrate inactivation of Escherichia coli thymidylate synthase (Asn177→ASP)

“…However, in several ternary complexes, with both a nucleotide and a folate derivative bound, the asymmetric unit of crystalline E. coli TS is the dimer [3][4][5]. TS undergoes a considerable conformational change upon ternary complex formation [3,21]. The root mean square (rms) difference is 0.9 A between equivalent a-carbon positions for E. coli TS in the unliganded state and in a ternary complex with dUMP and 10-(3-propynyl)-5,8-dideazafolate [3].…”