Characterization of the parameters affecting covalent binding stoichiometry in binary and ternary complexes of thymidylate synthase.
Covalent binding stoichiometries for both the enzyme:5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) binary complex and the enzyme:FdUMP:5,10-methylenetetrahydrofolate (inhibitory ternary) complex at equilibrium were measured by the trichloroacetic acid precipitation assay and shown to be a function of temperature, time, pH, salt concentration, buffer composition and thiol concentration. Incubation at 37 degrees C yielded the maximum covalent binding ratio (mol FdUMP/mol enzyme) for the latter binary (0.7) and ternary (1.7) complexes. In most buffers studied, the maximum covalent binding ratio (1.5-1.7) for the inhibitory ternary complex occurred over a broad pH range (4.5-8.0), while the optimum covalent binding ratio for binary complex was observed at a much narrower region centered between pH 5.5-6.5. In the presence of increasing concentrations of phosphate buffer, the maximum binding ratio for the covalent binary complex decreased from 0.63 in the absence of phosphate to 0.1 in the presence of 225 mM phosphate, while that for the inhibitory ternary complex was unchanged. When a ternary complex was formed with enzyme, FdUMP and (+/-)-tetrahydrofolate in the absence of phosphate, the FdUMP:enzyme covalent binding ratio was 1.8, while in the presence of 75 mM phosphate, the binding ratio was only 1.0. When exogenous thiol was removed by centrifugal column chromatography, the maximum binding stoichiometry of the resulting inhibitory ternary complex was 1.7 and was independent of added thiol over a 2 h incubation period at 37 degrees C. When extensive dialysis at 5 degrees C was used to remove the thiol, the maximum binding stoichiometry of the resulting inhibitory ternary complex was found to be dependent on both the concentration of added thiol and the time of incubation at 37 degrees C and did not exceed a value of 1.0.
Cisneros
R J
RJ
Department of Chemistry, University of South Carolina, Columbia 29208.
Dunlap
R B
RB
D002021:Buffers; D005468:Fluorodeoxyuridylate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D011485:Protein Binding; D013763:Tetrahydrofolates / Q000378:metabolism; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000378:metabolism*
D002021:Buffers; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D005468:Fluorodeoxyuridylate; D013940:Thymidylate Synthase