Kinetics and thermodynamics of the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase

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Abstract
Thymidylate synthase (TS), 5-fluorodeoxyuridylate (FdUMP), and 5,10-methylenetetrahydrofolate (CH2-H4folate) form a covalent complex in which a Cys thiol of TS is attached to the 6-position of FdUMP and the one-carbon unit of the cofactor is attached to the 5-position. The kinetics of formation of this covalent complex have been determined at several temperatures by semirapid quench methods. Together with previously reported data the results permit calculation of every rate and equilibrium constant in the interaction. Conversion of the noncovalent ternary complex to the corresponding covalent complex proceeds at a rate of 0.6 s-1 at 25.degree. C, and the dissociation constant for loss of CH2-H4 folate from the noncovalent ternary complex is .apprx. 1 .mu.M. Activation parameters for the formation of the covalent complex were shown to be Ea = 20 kcal/mol, .DELTA.G+ = 17.9 kcal/mol, .DELTA.H.dbldag. = 19.3 kcal/mol, and .DELTA.S.dbldag. = 0.005 kcal/(mol-deg). The equilibrium constant between the noncovalent and covalent ternary complexes is .apprx. 2 .times. 104, and the overall dissociation constant of CH2-H4folate from the covalent complex is .apprx. 10-11 M. The conversion of the noncovalent ternary complex to the covalent adduct is about 12-fold slower than kcat in the normal enzymic reaction. However, because the dissociation constant for CH2-H4folate from the noncovalent ternary complex is about 10-fold lower than that from the TS-dUMP-CH2-H4folate Michaelis complex, the terms corresponding to kcat/Km are nearly equal. We propose that some of the intrinsic binding energy of CH2-H4folate may be used to facilitate formation of a 5-iminium ion intermediate. We suggest that iminium ion formation is catalyzed by (a) general-acid catalysis at N-10 of CH2-H4folate and (b) enzyme-induced perturbations of the five-membered ring of the cofactor within the noncovalent TS-FdUMP-CH2-H4folate complex. The latter may involve hydrogen bonding of the enzyme general-acid catalyst to N-10, perturbation of the p-aminobenzoic acid moiety of the cofactor, and strain on the five-membered ring of the cofactor.

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