DGMP binding

Abstract: The stabilization of dUMP, FdUMP, and dGMP binding to Escherichia coli thymidylate synthase (TS) in the presence and absence of a folate analogue inhibitor of TS, 1843U, was determined by differential scanning calorimetry. When the enzyme is thermally unfolded in the presence of dUMP, two separate temperature transitions are evident, although only one binding site/dimer was detected in equilibrium dialysis experiments. In the absence of dUMP, TS shows a major peak of unfolding at 45 degrees C with a shoulder at 47 degrees C. In the presence of increasing amounts of dUMP progressive changes in the size of each peak occur, each associated with a higher temperature of unfolding. At a ratio of dUMP/TS of 100, a major peak predominates with an unfolding temperature (Td) of 60 degrees C. FdUMP shows a similar profile, while dGMP does not alter the Td of the enzyme since dGMP alone does not bind to TS. Despite the fact that 1843U binds tightly to TS in the absence of nucleotide ligands [Dev, I. K., Dallas, W.S., Ferone, R., Hanlon, H., McKee, D.D., & Yates, B. B. (1994) J.Biol. Chem. 269, 1873-1882], it exhibits only a small effect on the Td profile of TS. However, when 1843U is present, in addition to the nucleotides (dUMP, FdUMP, or dGMP), a Td of 72 degrees C is achieved and the enthalpy of unfolding is increased by one-third. The stabilizing effect of substrate binding to TS by 1843U examined by thermodynamic parameters can be attributed to the considerable extra amount of free energy released on formation of the ternary complex of TS-1843U-nucleotide. The tightness of this complex is due to the stacking energy that results from Van der Waals contacts between the nucleotide purine or pyrimidine ring and the benzoquinazoline ring of 1843U [Weichsel, A., Montfort, W. R., Cieśla, J., & Maley, F. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3493-3497], which induces a local conformational change in the protein. This conformational change is associated with a significant positive entropy change, which suggests that water is expelled from the active site region.

Abstract: Plasmodium deoxyguanylate pathways are an attractive area of investigation for future metabolic and drug discovery studies due to their unusual substrate specificities. We investigated the energetic contribution to thymidylate kinase substrate binding, and the forces underlying ligand recognition. The binding constant varied from 8 × 104 M−1 at 290 K to 6 × 104 M−1 at 310 K for dGMP, and from 16 × 104 M−1 at 290 K to 4 × 104 M−1 at 310 K for TMP. ΔC p was estimated as −1.75 kJ mol−1 K−1 for TMP and +2 kJ mol−1 K−1 for dGMP. In comparison with TMP, the binding of dGMP to PfTMK produced less favorable enthalpy change, positive or favorable entropic contribution at lower temperature, positive heat capacity change, negative $$ \Updelta S_{\text{HE}}^{^\circ } $$ , positive ΔS other, higher total solvent-exposed surface area and more or less rigid body binding. These changes indicate unfavorable conditions for proper binding and lower conformational changes, and suboptimal structural reordering during dGMP binding.

Abstract: Plasmodium falciparum thymidylate kinase (PfTMK) is an essential enzyme for the growth of the organism because of its critical role in the de novo synthesis of deoxythymidine 5'-diphosphate (TDP), a precursor for TTP that is required for DNA replication and repair. The kinetics, thermodynamic parameters, and substrate binding properties of PfTMK for TMP, dGMP, ADP, and ATP were measured and characterized by steady-state kinetics and a combination of isothermal titration calorimetry, tryptophan fluorescence titration, and NMR. Mutational studies were performed to investigate residues that contribute to the unique ability of PfTMK to also utilize dGMP as a substrate. Isothermal titration calorimetry experiments revealed that dGMP binding exhibits a unique half-site binding mechanism. The occlusion of the empty site in the dGMP complex is supported by molecular mechanics calculations. Relaxation dispersion experiments show that the dGMP and enzyme complex is more dynamic at the dimer interface than the TMP complex on the μs-ms time scale. The unique properties of dGMP binding need to be considered in the design of guanosine-based PfTMK-specific inhibitors.

Abstract: Having oxaliplatin as archetype, several platinum complexes with a carbohydrate moiety resembling the cyclohexane-1,2-diamine ligand of oxaliplatin have been prepared. As leaving groups, the anionic ligands iodide, oxalate, and malonate were utilized, and for comparison purposes the chloro complex was employed. All compounds were characterized by elemental analysis, nuclear magnetic resonance spectroscopy, and electrospray mass spectrometry. The crystal structure of (SP-4-3)-diiodo(2,3-diamino-2,3-dideoxy-D-glucose-kappa(2)N,N')platinum(II) was determined by X-ray diffraction. The affinity toward dGMP was assayed by capillary electrophoresis, revealing that the chloro complex shows the highest reactivity, followed by the iodo complex. In contrast, the binding kinetics of the dicarboxylato complexes are slower, with the malonato complex being the least reactive. Reactivity to dGMP in the cell-free system correlates with cytotoxicity in two of four human cancer cell lines as determined by means of the MTT assay. In three of the four cell lines, the chloro and the malonato complex are the most and the least active of the carbohydrate-Pt complexes, respectively, with IC(50) values differing only by factors of up to 3.2. Cytotoxicity of the chloro complex is one to two orders of magnitude lower than that of oxaliplatin, but still comparable to that of carboplatin in two of the four cell lines.