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Abstract

Endogenous thymidylate synthases, isolated from tissues or cultured cells of the same specific origin, have been reported to show differing slow-binding inhibition patterns. These were reflected by biphasic or linear dependences of the inactivation rate on time and accompanied by differing inhibition parameters. Considering importance for chemotherapeutic drug resistance, a possibility was tested of thymidylate synthase inhibition to be affected by post-translational modification, e.g. phosphorylation, by comparing sensitivities to inhibition by each of two slow-binding inhibitors, 5-fluoro-dUMP and N4-hydroxy-dCMP, of two fractions of purified recombinant mouse enzyme preparation, phosphorylated and non-phosphorylated, separated by metal oxide/hydroxide affinity chromatography on Al(OH)3 beads. The modification, found to concern histidine residues and influence kinetic properties by lowering Vmax, altered with each inhibitor studied both the pattern of the dependence of the inactivation rate on time from linear to biphasic, as well as slow-binding inhibition parameters. Being present on only one subunit of at least a great majority of phosphorylated enzyme molecules, it probably introduced dimer asymmetry, causing the altered time dependence of inactivation rate pattern (biphasic with the phosphorylated enzyme) and resulting in asymmetric binding of each inhibitor studied. The latter is reflected by the ternary complexes, stable under denaturing conditions, formed by only the non-phosphorylated subunit of the phosphorylated enzyme with each of the two inhibitors and N5,10-methylenetetrahydrofolate. Inhibition of the phosphorylated enzyme by N4-hydroxy -dCMP was found strongly dependent on [Mg2+], the cations demonstrated previously to influence also activity of endogenous mouse TS isolated from tumour cells.

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... The active site in the mTS -N 4 -OH-dCMP -DHF structure was closed, with the catalytic Cys189 covalently bound to the C(6) atom of N 4 -OH-dCMP, as indicated by the continuous electron density and distance of 1.87 Å between the C(6) and Cys189 sulfur. In accord, N 4 -OH-dCMP binds in the presence of meTHF to the enzyme and the resulting complex is stable under condition of SDS electrophoresis (10). Thus, in view of the latter, the inhibitor apparently causes a unique effect of "uncoupling" of the two TS-catalyzed reactions of the one-carbon group transfer and reduction, allowing to reveal the enzyme's capacity to use THF as a cofactor reducing the pyrimidine ring C(5) in the absence of the 5-methylene group. ...
... The electrophoretic tests involved SDS and native PAGE. SDS electrophoresis in 10% polyacrylamide gel under denaturing conditions [17] allowed analysis of the reaction mixture, following 13 μM mTS (relative to the monomer) protein incubation for 15 min at 37°C with 150 μM FdUMP and 750 μM THF [10]. Gels were fixed by incubation in 50 mM Tris-HCl, pH 8.0, containing 25% (v/v) 2-propanol [18]. ...
... Gels were fixed by incubation in 50 mM Tris-HCl, pH 8.0, containing 25% (v/v) 2-propanol [18]. Each gel was stained with Pro-Q® Diamond Phosphoprotein Gel Stain for phosphate groups and SYPRO® Ruby Protein Gel Stain (Molecular Probes) for protein, as previously described [10]. Comparison of the protein gel patterns, obtained with the complete and control (lacking the corresponding nucleotide, THF or both) reaction mixtures, showed gel shift resulting from the nucleotide covalent (withstanding condition of SDS electrophoresis) binding by the enzyme, as previously described [10]. ...
Article
In view of previous crystallographic studies, N4-hydroxy-dCMP, a slow-binding thymidylate synthase inhibitor apparently caused "uncoupling" of the two thymidylate synthase-catalyzed reactions, including the N5,10-methylenetetrahydrofolate one-carbon group transfer and reduction, suggesting the enzyme's capacity to use tetrahydrofolate as a cofactor reducing the pyrimidine ring C(5) in the absence of the 5-methylene group. Testing the latter interpretation, a possibility was examined of a TS-catalyzed covalent self-modification/self-inactivation with certain pyrimidine deoxynucleotides, including 5-fluoro-dUMP and N4-hydroxy-dCMP, that would be promoted by tetrahydrofolate and accompanied with its parallel oxidation to dihydrofolate. Electrophoretic analysis showed mouse recombinant TS protein to form, in the presence of tetrahydrofolate, a covalently bound, electrophoretically separable 5-fluoro-dUMP-thymidylate synthase complex, similar to that produced in the presence of N5,10-methylenetetrahydrofolate. Further studies of the mouse enzyme binding with 5-fluoro-dUMP/N4-hydroxy-dCMP by TCA precipitation of the complex on filter paper showed it to be tetrahydrofolate-promoted, as well as to depend on both time in the range of minutes and the enzyme molecular activity, indicating thymidylate synthase-catalyzed reaction to be responsible for it. Furthermore, the tetrahydrofolate- and time-dependent, covalent binding by thymidylate synthase of each 5-fluoro-dUMP and N4-hydroxy-dCMP was shown to be accompanied by the enzyme inactivation, as well as spectrophotometrically confirmed dihydrofolate production, the latter demonstrated to depend on the reaction time, thymidylate synthase activity and temperature of the incubation mixture, further documenting its catalytic character.
... This was overexpressed and purified as described previously [8,9]. The enzyme was judged to be near homogeneous by denaturing polyacrylamide slab gel electrophoresis using a 40, 20 and 10 lg sample. ...
... In view of the foregoing conclusion of obvious interest is potential functional consequence of this mechanism, as well as possibility to find structural features enabling the inactive conformation stabilization (arginine residue in a position homologous to hTS 163) in other TS structures. Considering strong influences of posttranslational modifications on kinetics and inhibition of TS-catalyzed reaction [9,26,27] and resulting wide variability of properties of the enzyme preparations of the same specific origin [2,9,27], a simple comparison of mTS and hTS properties would not answer the former question. However, Gibson et al. [28] obtained a mutant R163K of the human enzyme and found its catalytic activity to be higher than that of the parental hTS. ...
... In view of the foregoing conclusion of obvious interest is potential functional consequence of this mechanism, as well as possibility to find structural features enabling the inactive conformation stabilization (arginine residue in a position homologous to hTS 163) in other TS structures. Considering strong influences of posttranslational modifications on kinetics and inhibition of TS-catalyzed reaction [9,26,27] and resulting wide variability of properties of the enzyme preparations of the same specific origin [2,9,27], a simple comparison of mTS and hTS properties would not answer the former question. However, Gibson et al. [28] obtained a mutant R163K of the human enzyme and found its catalytic activity to be higher than that of the parental hTS. ...
Article
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Crystal structures of mouse thymidylate synthase (mTS) in complexes with (1) sulfate anion, (2) 2′-deoxyuridine 5′-monophosphate (dUMP) and (3) 5-fluoro-dUMP (FdUMP) and N5,10-methylenetetrahydrofolate (meTHF) have been determined and deposited in Protein Data Bank under the accession codes 3IHI, 4E5O and 5FCT, respectively. The structures show a strong overall similarity to the corresponding structures of rat and human thymidylate synthases (rTS and hTS, respectively). Unlike with hTS, whose unliganded and liganded forms assume different conformations (“inactive” and “active,” respectively) in the loop 181–197, in each of the three mTS structures, the loop 175–191, homologous to hTS loop 181–197, populates the active conformer, with catalytic Cys 189 buried in the active site and directed toward C(6) of the pyrimidine ring of dUMP/FdUMP, pointing to protein’s inability to adopt the inactive conformation. The binary structures of either dUMP- or sulfate-bound mTS, showing the enzyme with open active site and extended C-terminus, differ from the structure of the mTS–5-FdUMP–meTHF ternary complex, with the active site closed and C-terminus folded inward, thus covering the active site cleft. Another difference pertains to the conformation of the Arg44 side chain in the active site-flanking loop 41–47, forming strong hydrogen bonds with the dUMP/FdUMP phosphate moiety in each of the two liganded mTS structures, but turning away from the active site entrance and loosing the possibility of H-bonding with sulfate in the sulfate-bound mTS structure.
... The in vitro CK2mediated phosphorylation of TS was shown by Frączyk et al. Frączyk et al., 2015) and Ludwiczak et al., 2016. The phosphorylation lowered TS catalytic activity and affected binding of the TS inhibitor, 5-fluorodeoxyuridylate (FdUMP). ...
... There are reports indicating the occurrence of CK2-mediated phosphorylation of TS, DHFR, and SHMT Frączyk et al., 2015;Ludwiczak et al., 2016;Rusin et al., 2017;Skierka et al., 2019); however, to this date, there have been no studies showing the physiological role of CK2-mediated phosphorylation of thymidylate synthesis cycle enzymes. Our previous and present qPCR results indicate that inhibition of CK2 in CCRF-CEM cells treated with a specific CX-4945 inhibitor does not significantly influence the mRNA levels. ...
Article
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Thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT) constitute the thymidylate synthesis cycle providing thymidylate for DNA synthesis and repair. Our previous studies indicated that TS and DHFR are the substrates of protein kinase CK2. This work has been aimed at the elucidation of the effect of CK2 activity on cell cycle progression, thymidylate synthesis enzyme expression and localization, and the role of CK2-mediated TS phosphorylation in in vitro di- and trimolecular complex formation. The results were obtained by means of western blot, confocal microscopy, flow cytometry, quantitative polymerase chain reaction (QPCR), quartz crystal microbalance with dissipation monitoring (QCM-D), and microthermophoresis (MST). Our research indicates that CK2 inhibition does not change the levels of the transcripts; however, it affects the protein levels of DHFR and TS in both tested cell lines, i.e., A549 and CCRF-CEM, and the level of SHMT1 in CCRF-CEM cells. Moreover, we show that CK2-mediated phosphorylation of TS enables the protein (pTS) interaction with SHMT1 and leads to the stability of the tri-complex containing SHMT1, DHFR, and pTS. Our results suggest an important regulatory role of CK2-mediated phosphorylation for inter- and intracellular protein level of enzymes involved in the thymidylate biosynthesis cycle.
... (2015) (Frączyk et al., 2015). De manière intéressante, la phosphorylation de la TS affecte sa sensibilité à l'inactivation par le FdUMP (Cieśla et al., 2006;Ludwiczak et al., 2016). Ainsi la phosphorylation de certains résidus, dont la Ser 10 et Ser 16 , pourrait augmenter la résistance des cellules au 5-FU en empêchant l'inactivation de la TS par le FdUMP (Figures 22 et 23). ...
Thesis
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La O-GlcNAcylation (O-N-acétylglucosaminylation) est une MPT (modification post-traductionnelle) dynamique et réversible catalysée par un unique couple d’enzymes antagonistes : l’OGT (O-GlcNAc transférase) et l’OGA (O GlcNAcase). Elle est considérée comme un véritable senseur nutritionnel et régule un grand nombre de mécanismes cellulaires fondamentaux. En ciblant des oncoprotéines et des suppresseurs de tumeur, sa dérégulation est associée à la cancérogenèse et la progression tumorale. En revanche, son rôle dans la réponse aux thérapies anti-cancéreuses est très peu étudié. Il a été néanmoins montré récemment que l’hyper-O-GlcNAcylation impacte la réponse de certains cancers à des drogues telles que le tamoxifène, le cisplatine, le bortézomib et le 5-FU (5-fluorouracile). Le 5-FU est la chimiothérapie de référence du CCR (cancer colorectal) et la TS (Thymidylate Synthase) sa cible principale. La surexpression de la TS est un biomarqueur de résistance au 5-FU utilisé en clinique. La TS a été montrée comme étant O-GlcNAcylée mais le rôle de cette MPT n’a pas été élucidé. Il nous est donc paru intéressant d’analyser le « cross-talk » entre O-GlcNAcylation et réponse au 5-FU dans le CCR dans l’hypothèse que la O-GlcNAcylation pourrait impacter la sensibilité au 5-FU en régulant sa cible TS. Un modèle murin in vivo de CCR humains et des cellules coliques non cancéreuses et cancéreuses ont été utilisés pour analyser l’effet du 5-FU sur la O-GlcNAcylation globale des protéines et réciproquement l’impact de la O-GlcNAcylation sur le niveau et l’activité de la TS, et la réponse au 5-FU. Nos données in vitro corroborent nos résultats in vivo et soutiennent que le 5-FU diminue la O-GlcNAcylation globale et que, réciproquement, la O-GlcNAcylation augmente le niveau de TS et sensibilise le CCR au 5-FU. Nous avons déchiffré le mécanisme moléculaire sous-jacent mettant en lumière le rôle de la O-GlcNAcylation dans la stabilisation de la TS et sa protection contre la dégradation protéasomale. Deux sites de O-GlcNAcylation de la TS ont été identifiés : la Thr251 à l’interface de dimérisation de l’enzyme et la Thr306 dans la séquence dégron carboxy-terminale connue pour contrôler sa dégradation. Ensemble nos résultats proposent une nouvelle stratégie thérapeutique combinant le 5-FU à un inhibiteur de l’OGA afin d’améliorer la réponse du CCR à la chimiothérapie à base de 5-FU.
... The active site in the mTS-N 4 -OH-dCMP-DHF structure was closed, the catalytic Cys189 being covalently bound to the C(6) atom of N 4 -OH-dCMP, reflected by the continuous electron density and distance of 1.87 Å between the C(6) and Cys189 Sγ sulfur atom (Scheme 1). In accord, N 4 -OH-dCMP remained bound to the enzyme under conditions of the SDS electrophoresis [15]. Of note is that in a ternary mTS complex, formed in the presence of FdUMP and meTHF (PDB ID: 5FCT; Table 1), all three components were covalently bound [13], as expected based on the corresponding TS structures published previously [12]. ...
Article
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Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer.
... These observations can be understood in terms of a negative cooperativity between the two subunits [43,[60][61][62]. Nevertheless, a recent report [63] showed that for a recombinant mTS, time-and inhibitor-dependent inactivation relationship is linear (for both N 4 -OH-dCMP and FdUMP), in contrast to the wild-type enzymes from Mus musculus, thus attesting to the lack of the cooperativity. This changed, for both types of inhibitors, when the recombinant mTS had been phosphorylated. ...
Article
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A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson–Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme–ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the ”abortive reaction” inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.
... It was reported that hsTSase could under particular circumstances have methylated glutamates and phosphorylation at serine. However, neither the modification sites nor the functional effect of those modifications, nor the conditions under which these forms are generated, have been unequivocally documented [14,16,17]. While a plethora of kinetic, structural and mechanistic studies are available on bacterial TSase [1,4,8,[18][19][20][21][22], mammalian TSase lacks such deep interrogations. ...
Article
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Thymidylate Synthase (TSase) is a highly conserved enzyme that catalyzes the production of the DNA building block thymidylate. Structurally, functionally and mechanistically, bacterial and mammalian TSases share remarkable similarities. Because of this closeness, bacterial enzymes have long been used as model systems for human TSase. Furthermore, while TSase inhibitors have long served as chemotherapeutic drugs, no TSase inhibitor serves as an antibiotic. Despite their high resemblance, the mammalian TSases are distinct in a few known aspects, such as having a N-terminal tail and two insertions in the primary sequence and active/inactive conformations. Here, we aim to comprehensively characterize human (hs) TSase and delineate its contrasts and the similarities to the well-studied Escherichia coli (ec) TSase. We found that, in contrast to ecTSase, Mg²⁺ does not enhance reaction rates for hsTSase. The temperature dependence of intrinsic kinetic isotope effects (KIEs), on the other hand, suggests that Mg²⁺ has little or no impact on the transition state of hydride transfer in either enzyme, and that the transition state for the hydride transfer in hsTSase is looser than in ecTSase. Additionally, the substrates’ binding order is strictly ordered for ecTSase but slightly less ordered for hsTSase. The observed kinetic and functional differences between bacterial and human enzymes may aid in the development of antibiotic drugs with reduced toxicity.
... C. elegans TS was expressed and purified as described earlier [18]. With both TS proteins phosphatase inhibitors were present in all purification buffers as described earlier [64]. TS activity was monitored as previously described [16]. ...
Article
Dihydropyrimidine dehydrogenase (DPD) catalyzes the two-electron reduction of pyrimidine bases uracil and thymine as the first step in pyrimidine catabolism. The enzyme achieves this simple chemistry using a complex cofactor set including two flavins and four Fe4S4 centers. The flavins, FAD and FMN, interact with respective NADPH and pyrimidine substrates and the iron-sulfur centers form an electron transfer wire that links the two active sites that are separated by 56 Å. DPD accepts the common antineoplastic agent 5-fluorouracil as a substrate and so undermines the establishment of efficacious toxicity. Though studied for multiple decades, a precise description of the behavior of the enzyme had remained elusive. It was recently shown that the active form of DPD has the cofactor set of FAD-4(Fe4S4)-FMNH2. This two-electron reduced state is consistent with fewer mechanistic possibilities and data suggests that the instigating and rate determining step in the catalytic cycle is reduction of the pyrimidine substrate that is followed by relatively rapid oxidation of NADPH at the FAD that, via the electron conduit of the 4(Fe4S4) centers, reinstates the FMNH2 cofactor for subsequent catalytic turnover.
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Dihydropyrimidine dehydrogenase (DPD) catalyzes the initial step in the catabolism of the pyrimidines uracil and thymine. Crystal structures have revealed an elaborate subunit architecture consisting of two flavin cofactors, apparently linked by four Fe4S4 centers. Analysis of the DPD reaction(s) equilibrium position under anaerobic conditions revealed a reaction that favors dihydropyrimidine formation. Single-turnover analysis shows biphasic kinetics. The serine variant of the candidate general acid, cysteine 671, provided enhanced kinetic resolution for these phases. In the first event, one subunit of the DPD dimer takes up two electrons from NADPH in a reductive activation step. Spectrophotometric deconvolution suggests that thes electrons reside on one of the two flavins. That oxidation of the enzyme by dioxygen can be suppressed by the addition of pyrimidine, is consistent with these electrons residing on the FMN. The second phase involves further oxidation of NADPH and concomitant reduction of the pyrimidine substrate. During this phase no net reduction of DPD cofactors is observed indicating that the entire cofactor set acts as a wire, transmitting electrons from NADPH to the pyrimidine rapidly. This indicates that the availability of the proton from C671 general acid controls the transmittance of electrons from NADPH to the pyrimidine. Acid quench and HPLC product analysis of single-turnover reactions with limiting NADPH confirmed 2:1, NADPH:pyrimidine stoichiometry for the enzyme accounting for successive activation and pyrimidine reduction. These data support an alternating subunit model in which one protomer is activated and turns over before the other subunit can be activated and enter catalysis.
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Enzyme motions on a broad range of time scales can play an important role in various intra- and intermolecular events, including substrate binding, catalysis of the chemical conversion, and product release. The relationship between protein motions and catalytic activity is of contemporary interest in enzymology. To understand the factors influencing the rates of enzyme-catalyzed reactions, the dynamics of the protein-solvent-ligand complex must be considered. The current review presents two case studies of enzymes-dihydrofolate reductase (DHFR) and thymidylate synthase (TSase)-and discusses the role of protein motions in their catalyzed reactions. Specifically, we will discuss the utility of kinetic isotope effects (KIEs) and their temperature dependence as tools in probing such phenomena.
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To solve the inhibition mechanism of thymidylate synthase (TS) by N4-hydroxy-dCMP (N4-OH-dCMP), crystallographic studies were undertaken. Structures of three mouse TS (mTS) complexes with the inhibitor were solved, based on crystals formed by the enzyme protein in the presence of either only N4-OH-dCMP [crystal A, belonging to the space group C 1 2 1, with two monomers in asymmetric unit (ASU), measured to 1.75 Å resolution] or both N 4-OH-dCMP and N5,10-methylenetetrahydrofolate (mTHF) (crystals B and C, both belonging to the space group C 2 2 21, each with a single monomer in ASU, measured to resolution of 1.35 Å and 1.17 Å , respectively). Whereas crystal A-based structure revealed the mTS-N4-OH-dCMP binary complex, as expected, crystals B- and C-based structures showed the enzyme to be involved in a ternary complex with N4-OH-dCMP and noncovalently bound dihydrofolate (DHF), instead of expected mTHF, suggesting the inhibition to be a consequence of an abortive enzyme-catalyzed reaction, involving a transfer of the onecarbon group to a hitherto unknown site and oxidation of THF to DHF. Moreover, both C(5) and C(6) inhibitor atoms showed sp3 hybridization, suggesting C(5) reduction, with no apparent indication of C(5) proton release. In accordance with our previous results, in all subunits of these structures the inhibitor molecule was identified as the anti rotamer of imino tautomer, forming, similar to deoxyuridine monophosphate, two hydrogen bonds with a conservative asparagine (mouse Asn220) side chain.
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Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.
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A line of human lymphocytic leukemia cells (CCRF-CEM) has been obtained which is 140-fold resistant to the potent cell growth inhibitor 5-fluoro-2'-deoxyuridine (FdUrd). The cells were also 11-fold cross-resistant to 5-fluorouracil. In contrast to several previous studies involving FdUrd-resistant mouse cells, thymidylate synthetase levels were not substantially elevated in these FdUrd-resistant human leukemic cells. Thymidine kinase activity was also unchanged in the resistant cells, although the levels of 5-fluoro-2'-deoxyuridylate (FdUMP), the potent inhibitor of thymidylate synthetase, generated at equimolar doses of FdUrd were about 40% lower than in the sensitive cells. Studies of the kinetics of FdUMP binding to thymidylate synthetase isolated from the FdUrd-resistant cells disclosed a considerably higher dissociation constant (Kd = 1.0 X 10(-9) M) for the ternary covalent enzyme . FdUMP . 5,10-methylene tetrahydrofolate complex compared to the value obtained with enzyme from sensitive cells (Kd = 4.4 X 10(-11) M). The thymidylate synthetase from the FdUrd-resistant cells also showed 17-fold weaker binding of 2'-deoxyuridylate, even though the Km value for 2'-deoxyuridylate was 3-fold lower compared to the enzyme from FdUrd-sensitive cells. The turnover number of the altered enzyme was 1.8-fold higher than that for the normal enzyme but the rate constants for the release of FdUMP from the ternary complex, which is also an enzyme-catalyzed reaction, were identical for both enzymes. Electrophoresis of the radiolabeled ternary complexes on nondenaturing gels showed small but reproducible differences in migration rates. These results demonstrate that the mechanism of resistance to FdUrd in this cell line involves an alteration in the target enzyme, thymidylate synthetase, which causes it have a lower affinity for nucleotides.
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Thymidylate synthase (TS) catalyzes methylation of dUMP to dTMP and is the target of cancer chemotherapeutic agents (e.g. 5-fluorouracil). Here, we used error-prone PCR to mutagenize the full-length human TS cDNA and then selected mutants resistant to 5-fluorodeoxyuridine in a bacterial complementation system. We found that resistant mutants contained 1-5 amino acid substitutions and that these substitutions were located along the entire length of the polypeptide. Mutations were frequent near the active site Cys(195) and in the catalytically important Arg(50) loop; however, many mutations were also distributed throughout the remainder of the cDNA. Mutants containing a single amino acid replacement identified the following 14 residues as unreported sites of resistance: Glu(23), Thr(51), Thr(53), Val(84), Lys(93), Asp(110), Asp(116), Pro(194), Ser(206), Met(219), His(250), Asp(254), Tyr(258), and Lys(284). Many of these residues are distant from the active site and/or have no documented function in catalysis or resistance. We conclude that mutations distributed throughout the linear sequence and three-dimensional structure of human TS can confer resistance to 5-fluorodeoxyuridine. Our findings imply that long range interactions within proteins affect catalysis at the active site and that mutations at a distance can yield variant proteins with desired properties.
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Mouse thymidylate synthase R209K (a mutation corresponding to R218K in Lactobacillus casei), overexpressed in thymidylate synthase-deficient Escherichia coli strain, was poorly soluble and with only feeble enzyme activity. The mutated protein, incubated with FdUMP and N(5,10)-methylenetetrahydrofolate, did not form a complex stable under conditions of SDS/polyacrylamide gel electrophoresis. The reaction catalyzed by the R209K enzyme (studied in a crude extract), compared to that catalyzed by purified wild-type recombinant mouse thymidylate synthase, showed the K(m) value for dUMP 571-fold higher and V(max) value over 50-fold (assuming that the mutated enzyme constituted 20% of total crude extract protein) lower. Thus the ratios k(cat, R209K)/k(cat, 'wild') and (k(cat, R209K)/K(m, R209K)(dUMP))/( k(cat, 'wild')/K(m, 'wild')(dUMP)) were 0.019 and 0.000032, respectively, documenting that mouse thymidylate synthase R209, similar to the corresponding L. casei R218, is essential for both dUMP binding and enzyme reaction.
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Crude extract specific activities of thymidylate synthase, dUTPase, thymidine kinase and dihydrofolate reductase were high during the development of Caenorhabditis elegans, the dauer larva activities being similar to those previously determined in Trichinella spiralis and T. pseudospiralis muscle larvae (with the exception of thymidine kinase, not detected in Trichinella). High thymidylate synthase expression in developmentally arrested larvae, demonstrated also at the mRNA and protein levels, is in agreement with a global cell cycle arrest of dauer larvae and indicates this unusual cell cycle regulation pattern can be shared by developmentally arrested larvae of C. elegans and the two Trichnella species. Hence, the phenomenon may be characteristic for developmentally arrested larvae of different nematodes, rather than specific for the parasitic Trichinella muscle larvae. Endogenous C. elegans thymidylate synthase was purified and its molecular properties compared with those of the recombinant protein, expression of the latter in E. coli cells confirming the NCBI database sequence identity.
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Our current understanding of the mechanisms of action of antitumor agents and the precise mechanisms underlying drug resistance is that these two processes are directly linked. Moreover, it is often possible to delineate chemoresistance mechanisms based on the specific mechanism of action of a given anticancer drug. A more holistic approach to the chemoresistance problem suggests that entire metabolic pathways, rather than single enzyme targets may better explain and educate us about the complexity of the cellular responses upon cytotoxic drug administration. Drugs, which target thymidylate synthase and folate-dependent enzymes, represent an important therapeutic arm in the treatment of various human malignancies. However, prolonged patient treatment often provokes drug resistance phenomena that render the chemotherapeutic treatment highly ineffective. Hence, strategies to overcome drug resistance are primarily designed to achieve either enhanced intracellular drug accumulation, to avoid the upregulation of folate-dependent enzymes, and to circumvent the impairment of DNA repair enzymes which are also responsible for cross-resistance to various anticancer drugs. The current clinical practice based on drug combination therapeutic regimens represents the most effective approach to counteract drug resistance. In the current paper, we review the molecular aspects of the activity of TS-targeting drugs and describe how such mechanisms are related to the emergence of clinical drug resistance. We also discuss the current possibilities to overcome drug resistance by using a molecular mechanistic approach based on medicinal chemistry methods focusing on rational structural modifications of novel antitumor agents. This paper also focuses on the importance of the modulation of metabolic pathways upon drug administration, their analysis and the assessment of their putative roles in the networks involved using a meta-analysis approach. The present review describes the main pathways that are modulated by TS-targeting anticancer drugs starting from the description of the normal functioning of the folate metabolic pathway, through the protein modulation occurring upon drug delivery to cultured tumor cells as well as cancer patients, finally describing how the pathways are modulated by drug resistance development. The data collected are then analyzed using network/netwire connecting methods in order to provide a wider view of the pathways involved and of the importance of such information in identifying additional proteins that could serve as novel druggable targets for efficacious cancer therapy.
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Thymidylate synthase (TSase) is a clinically important enzyme because it catalyzes synthesis of the sole de novo source of deoxy-thymidylate. Without this enzyme, cells die a "thymineless death" since they are starved of a crucial DNA synthesis precursor. As a drug target, TSase is well studied in terms of its structure and reaction mechanism. An interesting mechanistic feature of dimeric TSase is that it is "half-the-sites reactive", which is a form of negative cooperativity. Yet, the basis for this is not well-understood. Some experiments point to cooperativity at the binding steps of the reaction cycle as being responsible for the phenomenon, but the literature contains conflicting reports. Here we use ITC and NMR to resolve these inconsistencies. This first detailed thermodynamic dissection of multi-site binding of dUMP to E. coli TSase shows the nucleotide binds to the free and singly bound forms of the enzyme with nearly equal affinity over a broad range of temperatures and in multiple buffers. While small but significant differences in ΔC°P for the two binding events show that the active sites are not formally equivalent, there is little-to-no allostery at the level of ΔG°bind. In addition NMR titration data reveal that there is minor inter-subunit cooperativity in formation of a ternary complex with the mechanism based inhibitor, 5F-dUMP, and cofactor. Taken together, the data show that functional communication between subunits is minimal for both binding steps of the reaction coordinate.
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Over the past 60 years, chemotherapeutic agents that target thymidylate biosynthesis and the enzyme thymidylate synthase (TS) have remained among the most-successful drugs used in the treatment of cancer. Fluoropyrimidines, such as 5-fluorouracil and capecitabine, and antifolates, such as methotrexate and pemetrexed, induce a state of thymidylate deficiency and imbalances in the nucleotide pool that impair DNA replication and repair. TS-targeted agents are used to treat numerous solid and haematological malignancies, either alone or as foundational therapeutics in combination treatment regimens. We overview the pivotal discoveries that led to the rational development of thymidylate biosynthesis as a chemotherapeutic target, and highlight the crucial contribution of these advances to driving and accelerating drug development in the earliest era of cancer chemotherapy. The function of TS as well as the mechanisms and consequences of inhibition of this enzyme by structurally diverse classes of drugs with distinct mechanisms of action are also discussed. In addition, breakthroughs relating to TS-targeted therapies that transformed the clinical landscape in some of the most-difficult-to-treat cancers, such as pancreatic, colorectal and non-small-cell lung cancer, are highlighted. Finally, new therapeutic agents and novel mechanism-based strategies that promise to further exploit the vulnerabilities and target resistance mechanisms within the thymidylate biosynthesis pathway are reviewed.
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Resistance to chemotherapy and molecularly targeted therapies is a major problem facing current cancer research. The mechanisms of resistance to 'classical' cytotoxic chemotherapeutics and to therapies that are designed to be selective for specific molecular targets share many features, such as alterations in the drug target, activation of prosurvival pathways and ineffective induction of cell death. With the increasing arsenal of anticancer agents, improving preclinical models and the advent of powerful high-throughput screening techniques, there are now unprecedented opportunities to understand and overcome drug resistance through the clinical assessment of rational therapeutic drug combinations and the use of predictive biomarkers to enable patient stratification.
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Owing to the absence of antiparasitic vaccines and the constant threat of drug resistance, the development of novel antiparasitic chemotherapies remains of major importance for disease control. A better understanding of drug transport (uptake and efflux), drug metabolism and the identification of drug targets, and mechanisms of drug resistance would facilitate the development of more effective therapies. Here, we focus on malaria and African trypanosomiasis. Wereview existing drugs and drug development, emphasizing high-throughput genomic and genetic approaches, which hold great promise for elucidating antiparasitic mechanisms. We describe the approaches and technologies that have been influential for each parasite and develop new ideas for future research directions, including mode-of-action studies for drug target deconvolution. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 54 is January 06, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
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Inhibitors of enzyme-catalysed reactions can be divided into four classes according to the rate and strength of their interactions with enzymes.
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It is known that the inhibition of thymidylate synthetase by the substrate analogue FdUMP results from the formation of a tightly bound ternary complex in which both FdUMP and the cofactor (5,10-CH2H4folate) are simultaneously bound to the enzyme. It is believed that the ternary complex results because FdUMP is able to function in a manner identical with dUMP during the initial stages of catalysis, but gets "stuck" partway through. The stage at which no further reaction occurs is postulated to be a proton abstraction which, in the case of FdUMP, would require an unfavorable C-F bond cleavage. In this work we investigated the structure of the ternary complex by 19F NMR to determine the structure of the proposed intermediates and to explore the possible mechanistic implications. The binding of FdUMP to form a ternary complex is accompanied by an 12.4-ppm shift toward increased shielding. With the aid of model compounds it is possible to interpret this shift to be the result of an attack at the pyrimidinyl 6 position by a nucleophile (presumably a cysteinyl SH) followed by attachment at the 5 position to the CH2 of the cofactor. Verification of the latter point was obtained indirectly by loss of H-F coupling (unresolved) and directly by observing a 13C-19F coupling constant when ternary complex was formed from cofactor prepared with CD2O and 13CD2O (90% 13C enriched), respectively. Denaturation of the ternary complex causes an 10.5-ppm shift of the 19F resonance toward decreased shielding. The ternary complex remains intact as evidenced by the retention of the 19F-13C coupling to the cofactor. By analogy to α-fluorocyclohexanone, this shift reveals that the C-F bond has moved relative to the plane of anisotropy of the adjacent carbonyl group, i.e., upon denaturation the pyrimidine ring undergoes a conformational change. A sharpening of the 19F resonance upon denaturation concurs with the greater mobility of the denatured complex. Indirect measurement of the 1H-19F coupling constants to both the CH2 of cofactor and H6 of the pyrimidine ring (using deuterium differencing) and application of the Karplus-type relationship enable a detailed representation of the relative spatial orientations of the groups on the pyrimidine 5,6 bond to be derived. In native ternary complex one proton of the methylene group of 5,10-methylenetetrahydrofolate is trans to the fluorine while the other is gauche. The proton at C-6 of the nucleotide and the fluorine are in a pseudo-trans-diequatorial relationship (i.e., the cysteine and the methylene group must be trans diaxial). Denaturation alters this arrangement such that the fluorine and the C-6 proton are trans diaxial.
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N{sup 4}-Hydroxy-dCMP (N{sup 4}-OH-dCMP), N{sup 4}-methoxy-dCMP (N{sup 4}-OMe-dCMP), and their 5-fluoro congeners were all slow-binding inhibitors of Ehrlich carcinoma thymidylate synthase (TS), competitive with respect to dUMP, and had differing kinetic constants describing interactions with the two TS binding sites. N{sup 4}-OH-dCMP was not a substrate and its inactivation of TS was methylenetetrahydrofolate-dependent, hence mechanism-based. K{sub i} values for N{sup 4}-OH-dCMP and its 5-fluoro analogue were in the range 10{sup {minus}7}-10{sup {minus}8} M, 2-3 orders of magnitude higher for the corresponding N{sup 4}-OMe analogues. The 5-methyl analogue of N{sup 4}-OHdCMP was 10{sup 4}-fold less potent, pointing to the anti rotamer of the imino form of exocyclic N{sup 4}-OH, relative to the ring N(3), as the active species. This is consistent with weaker slow-binding inhibition of the altered enzyme from 5-FdUrd-resistant, relative to parent, L1210 cells by both FdUMP and N{sup 4}-OH-dCMP, suggesting interaction of both N{sup 4}-OH and C(5)-F groups with the same region of the active center. Kinetic studies with purified enzyme from five sources, viz., Ehrlich carcinoma, L1210 parental, and 5-FdUrd-resistant cells, regenerating rat liver, and the tapeworm Hymenolepis diminuta, demonstrated that addition of a 5-fluoro substituent to N{sup 4}-OH-dCMP increased its affinity from 2- to 20-fold formore » the enzyme from different sources. With the Ehrlich and tapeworm enzymes, N{sup 4}-OH-FdCMP and FdUMP were almost equally effective inhibitors.« less
Article
The role of the pyrimidine N(3)-H in binding of dUMP derivatives to thymidylate synthase was evaluated with the aid of a new dUMP analogue, 5-fluoro-4-thio-dUMP, synthesized by an improved thiation and enzymatic phosphorylation. The interaction of this analogue, and of 5-FdUMP, with the enzyme, and the pH-dependence of these interactions, were compared. Both were slow-binding competitive inhibitors of the enzyme from Ehrlich carcinoma, L1210 and CCRF-CEM cells, with Ki an order of magnitude higher for 5-fluoro-4-thio-dUMP than for 5-FdUMP. With both nucleotides, as well as the parent nucleosides, enzyme inactivation increased as the pH was lowered from 8 to 6. Maximum inactivation with 5-FdUrd was at pH 7.0, and with 5-fluoro-4-thio-dUrd at pH 6.0, in agreement with the higher pKa for the N(3)-H dissociation of the former, and pointing to participation of the N(3)-H as a hydrogen donor in binding to the enzyme.
Article
The role of the phosphate moiety of dUMP, and some analogues, in their interaction with mammalian thymidylate synthase, has been investigated. Substrate and inhibitor activities, and the pH-dependence of these activities, of dUMP and 5-FdUMP, as well as analogues with modified phosphate groups, were compared. The methyl ester of dUMP was neither a substrate nor an inhibitor. By contrast, the methyl ester of 5-FdUMP was a slow-binding inhibitor of the enzyme from L1210, Ehrlich ascites carcinoma and CCRF-CEM cells, with Ki values in the micromolar range. Both 5-FdUrd and the newly synthesized 5'-methylphosphonate of 5-FdUrd were also slow-binding inhibitors of the Ehrlich carcinoma enzyme, but with Ki values in the millimolar range. The interaction of dUMP, 5-FdUMP, and the methyl ester of the latter decreased with increase in pH, whereas that of the 5'-methyl-phosphonate of 5-FdUrd remained unchanged. The results are discussed in relation to the role of the phosphate hydroxyls of dUMP in binding to the enzyme. 5-FdUMP and its analogues exhibited differing interactions with two binding sites on the enzyme molecule, consistent with cooperativity of binding. A convenient procedure is described for the synthesis of 5-fluoro-2'-deoxyuridine-5'-methylphosphonate, applicable also to the preparation of other 5'-methylphosphonate analogues.
Article
1.1. Mouse thymus thymidylate synthase has been purified to apparent electrophoretic homogeneity and compared with the enzyme from mouse tumour L1210 and Ehrlich ascites carcinoma cells.2.2. The enzyme is a dimer composed of 35,000 mol. wt monomers.3.3. Mouse thymus and tumour enzymes exhibit allosteric properties reflected by cooperative binding of both dUMP and 5-fluoro-dUMP.4.4. Activation energy for the reaction, catalyzed by thymidylate synthase from mouse tumour but not from mouse thymus, lowers at temperatures above 34† C, reflecting a change of rate-limiting step in dTMP formation.5.5. MgATP at millimolar concentrations inhibits mouse thymus enzyme.
Article
Intracellular total Mg2+ and free Mg2+ are compartmentalized between cell organelles and within the cytosol. Different values of [Mg2+]i in the cytosol of the same cell type were measured by various investigators. A main reason for the differences is the uncertainty of the dissociation constants used for the Mg furaptra complex in the fluorescence method and for MgATP when 31P NMR was employed. The more realistic KD values of Mg furaptra and MgATP measured under in situ conditions are higher than the KDs used by most investigators. The [Mg2+]is obtained and the KDs used by various authors were presented. The role of intracellular Mg2, in metabolic functions and the action of various effectors on [Mg2+]i and [Ca2+] was reviewed. Intracellular Mg2+ may have a permissive role supporting the effector-induced mechanisms that are mediated by Ca2+ as a second messenger.
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Antiviral drug resistance is an increasing concern in immunocompromised patient populations, where ongoing viral replication and prolonged drug exposure lead to the selection of resistant strains. Rapid diagnosis of resistance can be made by associating characteristic viral mutations with resistance to various drugs as determined by phenotypic assays. Management of drug resistance includes optimization of host factors and drug delivery, selection of alternative therapies based on knowledge of mechanisms of resistance, and the development of new antivirals. This article discusses drug resistance in herpesviruses and hepatitis B.
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Thymidylate synthase (TS) is a homodimeric enzyme with two equivalent active sites composed of residues from both subunits. Despite the structural symmetry of the enzyme, certain experimental results are consistent with half-the-sites activity, suggesting negative cooperativity between the active sites. To gain insight into the mechanism behind this phenomenon, we explore segmental motions of rat TS in the absence of ligands, with normal mode analysis as a tool. Using solvent accessible surface area of the active site pocket as a monitor of the degree of opening of the active sites, we classified the first 25 nontrivial normal modes, obtained from the web server of the program ElNémo, according to the behavior of the active sites. We found seven modes that open and close both sites symmetrically and nine that do so in an anticorrelated fashion. We characterized the motions of these modes by visual inspection and through measurement of distances between selected atoms lining the active site pockets. The segments that regulate access to the active site correspond to the loop containing R44, helix K, and a long loop containing residues 103–125, in agreement with a large body of crystallographic studies. These elements can be activated together or in isolation. There are more asymmetric modes than symmetric ones in the set we analyzed, probably accounting for the half-the-sites behavior of the enzyme. Three of the asymmetric modes result in changes at the dimer interface and indicate the endpoints of possible communication pathways between the active sites. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 549–559, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
Article
5-(alpha-Bromoacetyl)-2'-deoxyuridine 5'-phosphate (1) is an active-site-directed irreversible inhibitor of thymidylate synthetase from Lactobacillus casei. Analysis of the rate of inactivation of the enzyme in the presence of substrate confirmed the intermediate formation of a reversible enzyme-inhibitor complex.
Article
Structural changes in the macromolecular targets of pharmacological agents can result in alterations in the efficacy of these agents. In previous studies, we identified a variant structural form of thymidylate synthase (TS) that is associated with relative resistance to 5-fluoro-2'-deoxyuridine, in a human colonic tumor cell line. We now report on the use of DNA transfer techniques to examine directly the effects of each TS form on drug response. TS cDNA constructs, corresponding to the normal or variant TS mRNA, were expressed in Chinese hamster lung cells or in Escherichia coli, and response to 5-fluoro-2'-deoxyuridine was determined. We observed that expression of the variant TS, which differs from the normal form by a tyrosine to histidine substitution at residue 33, confers a 4-fold level of drug resistance in the mammalian cells, as well as in bacteria. The possible role of Tyr-33 in 5-fluoropyrimidine-mediated inhibition of TS is discussed.
Article
N4-Hydroxy-dCMP (N4-OH-dCMP), N4-methoxy-dCMP (N4-OMe-dCMP), and their 5-fluoro congeners (syntheses of which are described) were all slow-binding inhibitors of Ehrlich carcinoma thymidylate synthase (TS), competitive with respect to dUMP, and had differing kinetic constants describing interactions with the two TS binding sites. N4-OH-dCMP was not a substrate (no dihydrofolate produced; no tritium released with 5-3H-labeled molecule), and its inactivation of TS was methylenetetrahydrofolate-dependent, hence mechanism-based, with arrest of a step posterior to addition of cofactor and blocking abstraction of the C(5) hydrogen. Ki values for N4-OH-dCMP and its 5-fluoro analogue were in the range 10(-7) - 10(-8) M, 2-3 orders of magnitude higher for the corresponding N4-OMe analogues. The 5-methyl analogue of N4-OH-dCMP was 10(4)-fold less potent, pointing to the anti rotamer of the imino form of exocyclic N4-OH, relative to the ring N(3), as the active species. This is consistent with weaker slow-binding inhibition of the altered enzyme from 5-FdUrd-resistant, relative to parent, L1210 cells by both FdUMP and N4-OH-dCMP, suggesting interaction of both N4-OH and C(5)-F groups with the same region of the active center. Kinetic studies with purified enzyme from five sources, viz., Ehrlich carcinoma, L1210 parental, and 5-FdUrd-resistant cells, regenerating rat liver, and the tapeworm Hymenolepis diminuta, demonstrated that addition of a 5-fluoro substituent to N4-OH-dCMP increased its affinity from 2- to 20-fold for the enzyme from different sources.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
Article
Thymidylate synthetase from 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells was purified to a state close to electrophoretical homogeneity (sp. act. = 1.3 mumoles/min/mg protein) and studied in parallel with the homogeneous preparation of the enzyme from the parental Ehrlich ascites carcinoma cells. The enzyme from the resistant cells compared to that from the parental cells showed: (i) a higher turnover number (at least 91 against 31 min-1), (ii) a higher inhibition constant (19 against 1.9 nM) for FdUMP (a tight-binding inhibitor of both enzymes), (iii) a lower activation energy at temps above 36 degrees (1.37 against 2.59 kcal/mole), and (iv) a lower inhibition constant (26 against 108 microM) for dTMP, inhibiting both enzymes competitively vs dUMP.
Article
Magnesium ions variably influenced activity of highly purified thymidylate synthetase preparations from different mouse tumors, activating the enzyme from Ehrlich ascites carcinoma (EAC) cells and inhibiting the enzyme from L1210 and L5178Y cells and from 5-fluorodeoxyuridine (FdUrd)-resistant EAC cells. In the presence of Mg2+ in a concentration resulting in either maximum activation or inhibition (25-30 mM) the enzymes from both the sensitive and FdUrd-resistant EAC lines and L5178Y cells were activated by ATP. Under the same conditions of Mg2+ concentration ADP and AMP inhibited the enzyme from the parental but not from the FdUrd-resistant EAC cells.
Article
5-Fluorouracil and 5-fluorodeoxyuridine monophosphate levels were estimated in 75 isolates of Candida albicans to determine whether 5-fluorocytosine susceptibility could be ideally correlated with the intrafungal formation of both 5-fluorodeoxyuridine monophosphate and 5-fluorouridine triphosphate or a reciprocal formation of the two metabolites to prove the mechanism of 5-fluorocytosine activity. Using the results of four in vitro susceptibility tests, we separated isolates of C. albicans into susceptibility groups. For most strains, there was a positive correlation between the degree of 5-fluorocytosine susceptibility and the inhibition of biosynthesis of both RNA and DNA, incorporation of 5-fluorouracil into RNA, inhibition of ribosomal protein synthesis, and levels of 5-fluorodeoxyuridine monophosphate. However, in some strains with a similar degree of 5-fluorocytosine resistance, either reduced incorporation of 5-fluorouracil or reduced 5-fluorodeoxyuridine monophosphate levels occurred, suggesting that these two mechanisms are not necessarily linked to each other and that both may be responsible for 5-fluorocytosine activity.
Article
The interaction of dTMP synthetase with N4-hydroxy-2'-deoxycytidylate (N4-HOdCMP) has been investigated. With use of standard assay conditions, N4-HOd-CMP is a competitive inhibitor with an apparent Ki of 8.0 microM. Incubation of N4-HOdCMP with dTMP synthetase in the presence of 5,10-methylenetetrahydrofolate (CH2-H4folate) resulted in a rapid time-dependent inactivation of the enzyme which was not first order and the formation of complexes which could be isolated on nitrocellulose filter membranes. With use of radioactive ligands, the isolable native complex was shown to possess 2 mol of N4-HOdCMP and 2 mol of CH2-H4folate/mol of dimeric enzyme; the apparent dissociation constant of N4-HOdCMP was 1.0 microM. Ultraviolet difference spectroscopy of the ternary complex showed a loss of the pyrimidine chromophore which did not reappear upon denaturation with NaDodSO4. The rate of dissociation of N4-HOdCMP from the ternary complex was biphasic in which one-half of the initially bound ligand dissociated with t 1/2 congruent to 2.3 min and the remainder with t 1/2 congruent to 13 min. When the N4-HOdCMP-CH2-H4folate-enzyme complex was denatured, one-half of the CH2-H4folate dissociated whereas all of the N4-HOdCMP remained bound to the enzyme. Taken together, our results indicate that N4-HOdCMP forms a covalent bond with dTMP synthetase and reveal an unusual asymmetry in the two subunits of the N4-HOdCMP-CH2-H4folate-enzyme complex. It appears that one subunit is covalently bound to N4-HOdCMP, which, in turn, is covalently linked to CH2-H4folate whereas the other subunit is covalently bound to N4-HOdCMP but CH2-H4folate is bound by noncovalent interactions.
Article
This chapter describes the techniques used in the detection and characterization of phosphoramidate-containing proteins. The more commonly employed assay procedures and isolation methods in the investigations of phosphoproteins involve the use of acid. Several techniques have been devised to detect, quantify, and characterize phosphorylated basic amino acids on proteins. When examining for acid-labile phosphorylation, care must be taken not to perform any procedure at low pH. Several methods have been employed that can detect acid-labile phosphorylation. The chapter outlines the methods that can be used as a general screening for acid-labile phosphates; they do not, however, indicate any specific acid-labile phosphoamino acid. There are numerous polyacrylamide gel electrophoretic systems that can be employed in the investigation of proteins containing acid-labile phosphate and in the determination of P–N and P–O content. Not only must the gel buffers be of neutral or basic pH, but the staining and destaining procedures must also adhere strictly to the absence of acid when dealing with phosphoramidate-containing proteins.
The main literature concerning the physiology and biochemistry as well as the pathophysiology and pathobiochemistry of magnesium is reviewed, including: Distribution and physico-chemical state of magnesium in the extracellular and intracellular fluid as well as in the subcellular organelles (membranes, mitochondria, microsomes, ribosomes). Intestinal resorption, transport across membranes and excretion by the kidney. Hormonal regulation of magnesium distribution and its clinical disturbances. Biochemical mechanism and the clinical effects of hypo- and hypermagnesemia.
Article
Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH2H4folate to produce dTMP and H2folate. Knowledge of the catalytic mechanism and structure of TS has increased substantially over recent years. Major advances were derived from crystal structures of TS bound to various ligands, the ability to overexpress TS in heterologous hosts, and the numerous mutants that have been prepared and analyzed. These advances, coupled with previous knowledge, have culminated in an in-depth understanding of many important molecular details of the reaction. We review aspects of TS catalysis that are most pertinent to understanding the current status of the structure and catalytic mechanism of the enzyme. Included is a discussion of available sources and assays for TS, a description of the enzyme's chemical mechanism and crystal structure, and a summary of data obtained from mutagenesis experiments.
Article
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 were < 1.0, 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.
Article
To determine how 5-fluoro-dUMP modifications may affect its specificity, 2-thio-5-fluoro-dUMP and 4-thio-5-fluoro-dUMP were compared as inhibitors of thymidylate synthases isolated from parental and FdUrd-resistant mouse leukemia L1210 cells, human and rat colon adenocarcinomas, regenerating rat liver and the tapeworm, Hymenolepis diminuta, differing in sensitivity to time- and N5,10-methylenetetrahydrofolate-dependent inactivation by 5-fluoro-dUMP (Ki values ranging from 10(-9) to 10(-7) M). Inactivation by 2-thio-5-fluoro-dUMP, relative to 5-fluoro-dUMP, was 5-20-fold weaker, with specificity for inactivation of different thymidylate synthases paralleling that of 5-fluoro-dUMP. By contrast, 4-thio-5-fluoro-dUMP showed very different specificity, being as potent an inactivator for some enzymes as 5-fluoro-dUMP, but 45-85-fold weaker for others. The results suggest that an interplay between substituents at C(4) and C(5) of the pyrimidine ring may affect the specificity of thymidylate synthase inactivation.
Article
A major mechanism underlying the cytotoxicity of fluoropyrimidine analogs such as 5-fluorouracil and 5-fluoro-2'-deoxyuridine (FdUrd) occurs via the formation of 5-fluoro-2'-deoxyuridylate (FdUMP), a tight-binding inhibitor of thymidylate synthase (TS). Genetic variation in the structure of the TS molecule is an important determinant of response to fluoropyrimidines, because such variation may affect the binding of FdUMP to the enzyme. Previous studies have shown that the colonic tumor cell line HCT116 expresses two structurally distinct TS polypeptides that differ by the presence of tyrosine or histidine at residue 33. Compared with the Tyr-33 form, the His-33 form confers a 3-4-fold level of FdUrd resistance to cells; this was postulated to be derived from the reduced affinity of the enzyme for FdUMP and N5,N10-methylenetetrahydrofolate, ligands required for the formation of a stable inhibitory complex. In the present study, the Tyr-33 and His-33 forms have been purified to homogeneity, and their properties have been compared in detail. The Km values for dUMP and N5,N10-methylenetetrahydrofolate in the TS reaction were not significantly different between the two enzymes. In contrast, the catalytic efficiency (kcat) was 8-fold lower for the His-33 form. Kinetic and equilibrium binding measurements demonstrated that the dissociation constant for FdUMP binding into the ternary complex was 3-4-fold higher for the His-33 form; this was shown to be due to both a decrease in the rate of FdUMP association with the enzyme and an increase in the rate of FdUMP dissociation from the ternary complex. A TS form containing phenylalanine at residue 33 was created by site-directed mutagenesis and was shown to be very similar to the Tyr-33 enzyme with regard to kcat, pH/activity profile, and effect on FdUrd response. Thus, it is the presence of histidine at residue 33, rather than the absence of tyrosine, that is responsible for the alterations in catalytic and ligand-binding functions exhibited by the His-33 form. Possible mechanisms by which the histidine residue perturbs the structure of the TS active site are discussed.
Article
Thymidylate synthase specific activity was found to remain at a constant level in crude extracts from muscle larvae, isolated (1-15 months after infection) by pepsin-HCI digestion, as well as from adult worms of Trichinella spiralis. The enzyme was purified and its molecular (monomer mol. wt 35 kD) and kinetic (sequential mechanism with the K(m) values 3.1 and 19 microM for dUMP and N5,10-methylenetetrahydrofolate, respectively) properties determined. 5-Fluoro-dUMP was a competitive, slow-binding inhibitor of the parasite enzyme. N5,10-methylenetetrahydrofolate analogues 10-propargy1-5,8- dideazafolate (CB3717), ZD1694, BW1843U89, and AG337 were weaker inhibitors of the parasite than regenerating rat liver enzyme. Inhibition by 10-propargyl-5,8-dideazafolate was strengthened by an increasing number of glutamate residues. Thymidine kinase activity could not be detected in the muscle larvae crude extracts.
Article
Thymidylate synthase (TS), a half-the-sites reactive enzyme, catalyzes the final step in the de novo biosynthesis of deoxythymidine monophosphate, dTMP, required for DNA replication. The cocrystal structure of TS from Pneumocystis carinii (PcTS), a new drug target for an important pathogen, with its substrate, deoxyuridine monophosphate (dUMP), and a cofactor mimic, CB3717, was determined. The structure, solved at 2.6 A resolution, shows an asymmetric dimer with two molecules of the substrate dUMP bound yet only one molecule of cofactor analogue bound. The structural evidence reveals that upon binding cofactor analogue and forming a covalent bond from the nucleophilic cysteine to the substrate, dUMP, at one active site, PcTS undergoes a conformational change that renders the opposite monomer incapable of forming a covalent bond or binding a molecule of cofactor analogue. The communication pathway between the two active sites is evident, allowing a structural definition of the basis of half-the-sites reactivity for thymidylate synthase and providing an example of such a mechanism for other half-the-sites reactive enzymes.
Article
Thymidylate synthase, dihydrofolate reductase and dUTPase specific activities were found to remain at a high and constant level in crude extracts from adult worms of Trichinella spiralis, as well as from muscle larvae of both Trichinella spiralis (isolated 1-24 months after infection) and Trichinella pseudospiralis (isolated 5.5-13 months after infection). The results obtained with Trichinella pseudospiralis muscle larvae isolated with the use of pepsin did not differ from those obtained when pepsin was not used. No thymidine kinase activity could be detected in muscle larvae of either species and thymidine phosphorylase could be found only in T. pseudospiralis larvae isolated without the use of pepsin. Muscle larvae of both species contained orotidylate phosphoribosyl transferase activity, pointing to a possibility of 5-fluorouracil activation. Uridine phosphorylase, another enzyme involved in 5-fluorouracil anabolism, was also present in T. pseudospiralis muscle larvae. Results of comparative studies on inhibition of purified T. spiralis and rat thymidylate synthases by substrate (4-thio-5-fluoro-dUMP, 2-thio-5-fluoro-dCMP and N4-hydroxy-dCMP) and cofactor (ZD 9331) analogues indicated only dUMP analogues to show feeble selectivity towards the parasite enzyme. A hypothesis is discussed, assuming high expression of thymidylate synthase in muscle larvae to be connected with their cells being arrested in the cell cycle.
Article
Flucytosine (5-FC) is a synthetic antimycotic compound, first synthesized in 1957. It has no intrinsic antifungal capacity, but after it has been taken up by susceptible fungal cells, it is converted into 5-fluorouracil (5-FU), which is further converted to metabolites that inhibit fungal RNA and DNA synthesis. Monotherapy with 5-FC is limited because of the frequent development of resistance. In combination with amphotericin B, 5-FC can be used to treat severe systemic mycoses, such as cryptococcosis, candidosis, chromoblastomycosis and aspergillosis. Recently, 5-FC has been combined with newer azole antifungal agents; it also plays an important role in a new approach to the treatment of cancer. The severe side effects of 5-FC include hepatotoxicity and bone-marrow depression. In most patients, these side effects are concentration dependent, predictable, possibly avoidable with close monitoring to maintain 5-FC concentrations at <100 mg/L, and reversible with drug discontinuation or reduction of dose. 5-FC is well absorbed after oral administration, penetrates into body tissues well and is excreted mainly by the kidneys. In renal failure, major dose adjustments have to be made. The most important drug interaction of 5-FC occurs with concomitant administration of 5-FC and nephrotoxic drugs, especially amphotericin B. Owing to the crucial role of glomerular filtration in 5-FC elimination, drugs that impair this mechanism will decrease the elimination of 5-FC and thus prolong its half-life.
Article
Antibiotics--compounds that are literally 'against life'--are typically antibacterial drugs, interfering with some structure or process that is essential to bacterial growth or survival without harm to the eukaryotic host harbouring the infecting bacteria. We live in an era when antibiotic resistance has spread at an alarming rate and when dire predictions concerning the lack of effective antibacterial drugs occur with increasing frequency. In this context it is apposite to ask a few simple questions about these life-saving molecules. What are antibiotics? Where do they come from? How do they work? Why do they stop being effective? How do we find new antibiotics? And can we slow down the development of antibiotic-resistant superbugs?
Article
In a previous study we demonstrated that Escherichia coli thymidylate synthase activity could be restored completely by incubating basically inactive mutants of this enzyme at room temperature with R(126)E, another inactive mutant [Maley, F., Pedersen-Lane, J., and Changchien, L.-M. (1995) Biochemistry 34, 1469-1474]. Since only one of the enzyme's two subunits possessed a functional active site and the restoration of activity could be titrated to be equivalent to that of the wild-type enzyme's specific activity, it was proposed that thymidylate synthase was a half-of-the-sites activity enzyme. We now provide additional support for this thesis by presenting an in-depth analysis of some conditions affecting the restoration of enzyme activity. For this purpose, we employed two mutants with marginal thymidylate synthase activity, Y(94)A and R(126)E. The parameters that were examined included pH, concentration of protein, temperature, and urea concentration, all of which influenced the rate of activity restoration. It was found, surprisingly, that by maintaining the amount of each protein constant, while increasing the volume of solution, the rate and total activity restored was greatly enhanced. Increasing the pH from 6.0 to 9.0 markedly increased the rate at which the optimal activity was restored, as did increasing the temperature from 4 to 40 degrees C. A similar effect was obtained when the incubation of the mutants was conducted at 4 degrees C in the presence of 1.5 M urea, a temperature at which activity is restored extremely slowly. Raising the pH to 9.0 resulted in an almost instantaneous restoration of activity at 4 degrees C. The manner in which thymidylate synthase activity is restored from the mutants in the presence of varying concentrations of ethanol, ethylene glycol, and glycerol suggests that changes in subunit interaction and enzyme conformation are in part responsible for the observed differences. Most significantly, at solution levels of 10%, ethanol was found to activate, while ethylene glycol inhibited slightly and glycerol was somewhat more inhibitory. At a concentration of 20%, ethanol inhibited rather strikingly, ethylene glycol was slightly more inhibitory than at 10%, and glycerol was strongly inhibitory. Since the net result of these findings is the suggestion that the restoration of thymidylate synthase activity is due to a separation of the mutant dimers into their respective subunits, followed by their recombination to an active heterodimer, evidence for this phenomenon was sought by separating the recombined dimers using nondenaturating polyacrylamide gel electrophoresis. Sequence analysis of the isolated homo- and heterodimers clearly demonstrated that the active enzyme is a product of subunit exchange, one that is very efficient relative to the wild-type enzyme, which did not exchange subunits unless denatured.
Article
SUMMARY Various biochemical studies have been carried out in vitro with Ehrlich ascites car cinoma cells susceptible and resistant to fluorinated pyrimidines. There is no major difference between the two cell lines with respect to the utilization of formate or thy midine for DNA thymine biosynthesis, nor the incorporation of uracil or orotic acid into RNA uracil. The incorporation of formate-C'4 into DNA thymine is inhibited by fluorinated pyrimidines to a considerably greater extent in the susceptible than in the resistant cells. Although in the resistant cells there is a decreased conversion of 5- fluorouracil into 5-fluorouridylic acid and incorporation into RNA, the amount of 5-fluoro-@'-deoxyuridine-5'-monophosphate (FUDRP) produced was equivalent in the two types of cells. In a cell-free system it was shown that thymidylate synthetase oh tamed from the susceptible cells was inhibited by FUDRP, whereas the enzyme ob tamed from the resistant cells was not inhibited by FUDRP. Thus, the mechanism of resistance to fluoropyrimidines in this line of the Ehrlich ascites carcinoma appears to involve the alteration of the enzyme, thymidylate synthetase, in such manner that FUDRPisnotinhibitory intheresistantcells.
Article
A novel method termed metal oxide affinity chromatography (MOAC) of enriching for phosphorylated proteins and peptides based on the affinity of the phosphate group for Al(OH)(3) is presented here. When compared to commercial phosphoprotein-enrichment kits, this method is more selective, more cost effective and easily applicable to method optimization. The use of glutamic and aspartic acid in the loading buffer significantly enhances selectivity. Standard protein mixtures and complex Arabidopsis thaliana leaf protein extracts were tested for efficacy of enrichment. The method can be applied to proteins extracted using either mild or denaturing conditions. The same Al(OH)(3) material is suitable for the enrichment of phosphopeptides out of a tryptic digest of alpha-casein. Peptide phosphorylation was revealed by beta-elimination of phosphate groups. Enrichment and in vivo phosphorylation of A. thaliana leaf proteins were confirmed with Pro-Q diamond stain. Several of the phosphoprotein candidates that were identified by MS are known to be phosphorylated in vivo in other plant species.