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Thymidylate synthase (TS) is a homodimeric enzyme that catalyzes the reductive methylation of dUMP by N5,N10-methylene-5,6,7,8-tetrahydrofolic acid, to form dTMP. Inhibition of TS by the dUMP analog 5-fluoro-dUMP (FdUMP) occurs through the formation of a covalent ternary complex containing the nucleotide analog, N5,N10-methylene-5,6,7,8-tetrahydrofolic acid, and the enzyme; this complex is termed the inhibitory ternary complex (ITC). In the present report, the kinetics of FdUMP binding into an ITC with purified preparations of human TS were examined. Rapid chemical-quench techniques, as well as steady state binding methods, showed that the enzyme contains two distinct FdUMP binding sites with different affinities for the nucleotide analog. Binding to the first, or high affinity, site was rapid and reached a maximum stoichiometry of 1.0 mol of FdUMP/mol of dimer; binding to the second, or low affinity, site was much slower and reached a stoichiometry of 1.7 mol of FdUMP/mol of dimer. Rate constants for FdUMP binding to and dissociation from the ITC (kon and koff, respectively) were determined, as were equilibrium dissociation constants (Kd). A naturally occurring mutant form of TS, which contains a tyrosine to histidine substitution at residue 33 and renders cells relatively resistant to fluoropyrimidines, exhibited a lower affinity for FdUMP specifically at the second binding site, with little or no change at the first. Hill coefficients were1.0, with the His-33 enzyme having a significantly lower coefficient than the wild-type enzyme. The results, in total, indicate that the two FdUMP binding sites on the TS dimer are nonequivalent. We suggest that such nonequivalence may be due to negative cooperativity, where nucleotide binding to the first subunit elicits a conformational change that results in reduced affinity for ligand at the second subunit. This negative cooperativity may be stronger for the His-33 mutant. Thus, the relative fluoropyrimidine resistance conferred by the His-33 substitution may be due to enhanced negative cooperative effects on FdUMP binding into the ITC, thereby reducing the effectiveness of the pyrimidine analog as an inhibitor of thymidylate biosynthesis. |
