Structure of Human Thymidylate Synthase Suggests Advantages of Chemotherapy with Noncompetitive Inhibitors

Abstract

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms. The emergence of resistance to the treatment is often related to the increased levels of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding, a feedback regulatory mechanism, and/or to the increased stability to intracellular degradation of TS⅐drug complexes (versus unliganded TS). The active site loop of human TS (hTS) has a unique conformation resulted from a rotation by 180°relative to its orientation in bacterial TSs. In this conformation, the enzyme must be inactive, because the catalytic cysteine is no longer positioned in the ligandbinding pocket. The ordered solvent structure obtained from high resolution crystallographic data (2.0 Å) suggests that the inactive loop conformation promotes mRNA binding and intracellular degradation of the enzyme. This hypothesis is supported by fluorescence studies, which indicate that in solution both active and inactive forms of hTS are present. The binding of phosphate ion shifts the equilibrium toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active form. Thus, TS inhibition via stabilization of the inactive conformation should lead to less resistance than is observed with presently used drugs, which are analogs of its substrates, dUMP and CH 2 H 4 folate, and bind in the active site, promoting the active conformation. The presence of an extension at the N terminus of native hTS has no significant effect on kinetic properties or crystal structure. Thymidylate synthase (TS)1 catalyzes the reductive methylation of 2Ј-deoxyuridine 5Ј-monophosphate (dUMP) to thymidine 5Ј-monophosphate (dTMP), using the co-substrate, 5,10-methylenetetrahydrofolate (CH 2 H 4 folate) as a 1-carbon donor and reductant. The physical structures of bacterial TSs have been relatively well defined, and crystallographic data, in concert with data derived from kinetic, spectroscopic, and sitedirected mutagenesis studies, have led to a detailed understanding of the catalytic mechanism of these enzymes (1). In contrast, relatively few investigations of mammalian TS structure and catalysis have been conducted. The three-dimensional structure of the native human TS (hTS) has been reported previously (2). The data showed a surprising feature not observed in TSs from other sources: loop 181-197 containing the catalytic cysteine, Cys-195, was in an inactive conformation, rotated ϳ180°with respect to its orientation in bacterial TSs, with the sulfhydryl of Cys-195 over 10 Å from the location of sulfhydryls of corresponding cysteine residues in bacterial enzymes. Subsequent determination of the structure of a ternary inhibitory complex between closely related ratTS (rTS) and dUMP and Tomudex (3) has shown that the ligands bind to the enzyme in the active conformation. Recently, it was found that also in the hTS⅐dUMP⅐Tomudex complex hTS is in the active conformation (4). The inactive conformation has...

Cooperative Inhibition of Human Thymidylate Synthase by Mixtures of Active Site Binding and Allosteric Inhibitors,

“…2) support this hypothesis. PDPA induces an increase in hTS fluorescence in a similar manner as P i and the arguments linking this phenomenon to the loop 181-197 conformational switching (12) should apply here as well. The lack of similar effects for the mouse enzyme (Fig.…”

“…The crystal structure of hTS has been initially determined using crystals obtained at high ammonium sulfate concentrations (11,12). At these conditions the active-site loop 181-197 is in a conformation different from that observed in bacterial TS.…”

Role of Y94 in Proton and Hydride Transfers Catalyzed by Thymidylate Synthase

“…The fractional conversion f was calculated by: [2] Figure 2 presents an example of measured H/T KIEs as function of f. Since the KIE on the proton abstraction has not been measured before, it is important to demonstrate that the KIE is reproducible in a series of independent experiments and that there is no upward or downward trends in the KIE as function of f. * Figure 3 (25-28): [3] where T (V/K) H, obs and T (V/K) D, obs are the observed competitive H/T and D/T KIEs; k T /k H represents the reciprocal of k H /k T (intrinsic H/T KIE). Although the intrinsic H/T KIE is the only unknown in this equation, it cannot be solved analytically.…”

Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

“…Besides allowing for a full masking of the TS active site, formation of this ternary complex stabilizes the active dimeric form of the protein, making C180 not accessible. 19 Graphical analysis of the 3D structure of the active conformer (pdb entry 1hvy) indicate that the reactive C43-C43 0 residues are 27.5 Å apart. Fluorescein (F, donor) and tetramethylrhodamine (T, acceptor) were chosen as the fluorescent tags for two reasons: (i) due to the extensive F-emission/T-absorption spectral overlap, which corresponds to a Förster's critical distance, R 0 , twice as large as the C43-C43 0 distance, 20 F-to-T FRET efficiency, u ET , is expected to be close to unity in this system; indeed, assuming the interchromophore distance to coincide with the C43-C43 0 distance, r ¼ 27.5 Å , i.e., that linker effects are negligible (see the maleimide structures in Scheme 1), and that the probes may undergo isotropic motion, 21 with the well known relationship, 20 we calculate u ET ¼ 0.985; (ii) the commercially available maleimides of the two fluorophores easily bind to the accessible cysteines by exploiting the reactivity of such a functional group towards thiols, with which it forms stable thioether bonds.…”