Simple separation of tritiated water and [3H]deoxyuridine from [5-3H]deoxyuridine 5'-monophosphate in the thymidylate synthase assay

A simple micromethod was developed for the accurate measurement of the activity of dTMP synthase in rat liver crude extracts. The reaction product of dTMP synthase activity assay, i.e., tritiated water, generated by the release of tritium from carbon-5 of [5-3H]deoxyuridine 5'-monophosphate (dUMP), was separated simply by 100% KOH absorption from [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]dUMP during the enzyme reaction. Tritiated water was trapped in three droplets of 100% KOH deposited on the underside of the vessels' lids, while [3H]dUrd remained in the bottom of vessels after absorption of the substrate, [5-3H]dUMP, from the reaction mixture by charcoal treatment. Under standard assay conditions in the crude extract of rat liver, the specific activities of dTMP synthase and dUMP phosphatase were 0.092 +/- 0.002 and 0.351 +/- 0.013 nmol/h/mg protein, respectively. This method was also adapted for dTMP synthase assay in crude extracts of rat hepatoma 3924A. The major advantages of this procedure are the elimination of the phosphatase activity which interferes with the estimation of dTMP synthase activity in crude extracts, one-step separation of 3H2O, high sensitivity (with a limit of detection of 10 pmol of 3H2O production), high reproducibility (less than +/- 4.3%), and capability to measure activity in small amounts of sample (30-45 micrograms protein).     

A bifunctional dCTP deaminase-dUTP nucleotidohydrolase from the hyperthermophilic archaeon Methanocaldococcus jannaschii

By the sequential action of dCTP deaminase and dUTPase, dCTP is converted to dUMP, the precursor of thymidine nucleotides. In addition, dUTPase has an essential role as a safeguard against uracil incorporation in DNA. The putative dCTP deaminase (MJ0430) and dUTPase (MJ1102) from the hyperthermophilic archaeon Methanocaldococcus jannaschii were overproduced in Escherichia coli. Unexpectedly, we found the MJ0430 protein capable of both reactions, i.e. hydrolytic deamination of the cytosine ring and hydrolytic cleavage of the phosphoanhydride bond between the alpha- and beta-phosphates. When the reaction was followed by thin layer chromatography using [3H]dCTP as substrate, dUMP and not dUTP was identified as a reaction product. In the presence of unlabeled dUTP, which acted as an inhibitor, no label was transferred from [3H]dCTP to the pool of dUTP. This finding strongly suggests that the two consecutive steps of the reaction are tightly coupled within the enzyme. The hitherto unknown bifunctionality of the MJ0430 protein appears beneficial for the cells because the toxic intermediate dUTP is never released. The MJ0430 protein also catalyzed the hydrolysis of dUTP to dUMP but with a low affinity for the substrate (Km >100 micro m). According to limited proteolysis, the C-terminal residues constitute a flexible region. The other protein investigated, MJ1102, is a specific dUTPase with a Km for dUTP (0.4 micro m) comparable in magnitude with that found for previously characterized dUTPases. Its physiological function is probably to degrade dUTP derived from other reactions in nucleotide metabolism.     

The turnover of deoxyuridine triphosphate during the HeLa cell cycle

The synthesis and breakdown of deoxyuridine triphosphate (dUTP) was studied to determine whether a dUTP pool is present at any stage of the HeLa cell cycle. Although cell extracts were found to be capable of phosphorylating dUMP to dUTP, only minimal quantities of intracellular dUMP, dUDP or dUTP could be detected. When thymidylate synthetase was blocked with FUdR the dUMP pool increased but no substantial increase in dUDP or dUTP was seen. A powerful and specific dUTP nucleotidohydrolase (dUTPase, EC3.6.1.23) which hydrolyses dUTP to dUMP and PPi was detected. The activity of this enzyme as well as that of the dUTP synthesizing enzymes was low in G1, rose through S and G2 and reached a maximum just prior to cell division. Pulsing experiments with [5-³H]UdR and [¹⁴C]TdR suggest that the size of the dUTP pool is 1% of the dTTP pool.     

Improved measurement of thymidylate synthetase activity by a modified tritium-release assay

Accurate quantitation of thymidylate synthetase activity using a tritium-release assay is dependent upon measurement of only that tritium released from deoxy[5-³H]uridine monophosphate ([³H]dUMP) during the biosynthesis of thymidylate. Removal of remaining [³H]dUMP on completion of the assay by charcoal adsorption and correction for the nonenzymatic release of tritium are necessary. Although over 99% of [³H]dUMP is removed immediately following addition of charcoal, these studies demonstrate that sufficient [³H]dUMP can remain to prevent accurate measurement of low levels of thymidylate synthetase activity. By delaying measurement of radioactivity for at least 24 h following addition of charcoal, this problem is minimized. To account for nonenzymatic release of tritium, a blank containing enzyme extract with omission of $\text{±,}\text{l}\text{-5,10-}\text{methylenetetrahydrofolate}$ is demonstrated to be more effective than the commonly used blank in which water is substituted for enzyme extract. In samples containing 5-fluoro-2′-deoxyuridine monophosphate (FdUMP), a potent inhibitor of thymidylate synthetase activity, an alternative blank containing a high concentration of FdUMP (approximately 1mM) is useful in demonstrating a theoretical maximal or complete inhibition of thymidylate synthetase activity.     

Identification and partial characterization of rabbit brain deoxyuridine 5′-triphosphatase

Adult rabbit brain contains the enzymatic machinery to convert deoxyuridine to deoxyuridine triphosphate (dUTP). Although dUTP as dUMP can be readily incorporated into DNA in place of thymidine monophosphate, we detected no (3H)dUMP in newly synthesized (3H)DNA in adult rabbit brain after the intraventricular injection of (3H)deoxyuridine. Only (3H)thymidine was detected. The probable explanation for the lack of incorporation of uracil into adult rabbit brain DNA is the presence of a specific, high affinity dUTPase which converts dUTP to dUMP and PP. After homogenization and ammonium sulfate fractionation of adult rabbit brain (35 to 75% saturation), a high affinity, specific dUTPase was detected in the dialyzed enzyme preparation. The Km and Vmax of the dUTPase were 0.2 microM and 36 pmol/mg protein/min, respectively. No high affinity dUTPase activity was detectable in liver. In brain, another enzyme hydrolyzed dUTP and dTTP (NTPase( to their respective diphosphates. NTPase, unlike dUTPase, was not sensitive to heating at 65 degrees C for five minutes. Thus, brain, like other tissues, contains a high affinity, specific dUTPase presumably to "sanitize" the cells of dUTP and, thus, protect the integrity of newly synthesized DNA.     

Methionine synthase assay based on coupling with thymidylate synthase reaction

Methionine synthase reaction may be coupled with thymidylate synthase-catalysed tritium release from [5-3H]dUMP via non-enzymatic reaction of formaldehyde with tetrahydrofolate. A convenient and sensitive assay of methionine synthase activity, based on this principle, is described.     

Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from Campylobacter jejuni

The enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and PPi thus controlling the incorporation of uracil into DNA genomes. In Campylobacter jejuni dUTPase exhibits structural properties of dimeric proteins characteristic of protozoa of the Kinetoplastidae family. In the present study we perform a kinetic analysis of Campylobacter dUTPase using the continuous spectrophotometric method and show that the enzyme is highly specific for deoxyuridine nucleotides. The Michaelis-Menten constant for dUTP was 0.66 microM while the k(cat) was 12.3 s(- 1). dUDP was also efficiently hydrolysed although the specificity constant, k(cat)/K(m), was five fold lower than for dUTP. The reaction product and the non hydrolysable analogue alpha,beta imido dUDP are potent inhibitors of the enzyme while several analogues of dUMP with substituents at the 3'- and 5'-positions active against trimeric dUTPases, show poor inhibitory activity. Apparent structural and kinetic differences with other eukaryotic dUTPases suggest that the present enzyme might be exploited as a target for new drugs against campylobacteriosis.     

Uracil DNa-glycosylase from HeLa cells: general properties, substrate specificity and effect of uracil analogs.

Uracil-DNA glycosylase was partially purified from HeLa cells. Various substrates containing [3H]dUMP residues were prepared by nick-translation of calf thymus DNA. The standard substrate was double-stranded DNA with [3H]dUMP located internally in the chain. Compared to the release of uracil from this substrate, a 3-fold increase in the rate was seen with single-stranded DNA, and a 20-fold reduction in the rate was observed when the [3H]dUMP-residue was located at the 3'end. The rate of [3H]uracil release decreased progressively when one, two or three of the dNMP residues were replaced by the corresponding rNMP; in the extreme case when the substrate contained [3H]dUMP in addition to rCMP, rGMP, and rAMP, the rate of [3H]uracil release was less than 3% of that of the control. The enzyme was inhibited to the same extent by uracil and the uracil analogs 6-aminouracil and 5-azauracil, but very weakly, or not at all, by 5 other analogs. Our results suggest strongly that uracil-DNA glycosylase has a high degree of selectivity for uracil in dUMP residues located internally in DNA chains and that the recognition of the correct substrate also depends on the residues flanking dUMP being deoxyribonucleotides.     

Purification and properties of the deoxyuridine triphosphate nucleotidohydrolase enzyme derived from HeLa S3 cells. Comparison to a distinct dUTP nucleotidohydrolase induced in herpes simplex virus-infected HeLa S3 cells

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) (EC 3.6.1.23) derived from HeLa S3 cells has been purified to near homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of about 16,000 nmol of dUMP hydrolyzed per min/mg of protein. The dUTPase enzyme derived from HeLa S3 cells appears to be composed to two equal molecular mass subunits, each being about 22,500 daltons. Association of these subunits to produce a 45,000-dalton protein is promoted by MgCl2. In the presence of EDTA enzyme activity is abolished and the enzyme dissociates into its monomeric form. MgCl2 will completely reverse the inhibition caused by EDTA and promote subunit association. MnCl2 will also promote association of the dUTPase subunits. However, MnCl2 will not completely reverse inhibition by EDTA. In addition, purified dUTPase, extensively dialyzed to remove contaminating ions, is activated almost 2-fold by the addition of 5 mM MgCl2. In contrast, addition of 5 mM MnCl2 to the dialyzed enzyme preparation will cause more than a 50% decrease in enzyme activity. This data indicates that Mg2+ is the natural prosthetic group for this enzyme. The Km value of dUTP for the purified enzyme is 3 X 10(-6) M in the presence of MgCl2. The turnover number for this enzyme has been calculated to be 550 molecules of dUTP hydrolyzed per min under standard assay conditions. Infection of HeLa S3 cells with herpes simplex type 1 virus induces a distinct species of dUTPase. This new species of dUTPase has an isoelectric point of 8.0, compared to an isoelectric point in the range of 5.7 to 6.5 for the HeLa S3 dUTPase. Molecular weight determinations of this new species of dUTPase indicate that the native enzyme is monomeric with a molecular weight of about 35,000. The virally induced dUTPase is inhibited by EDTA and this inhibition is reversed by MgCl2. Unlike the HeLa S3 dUTPase, the virally induced enzyme does not appear to be composed of subunits.     

Thymidylate synthase activity in the development of Hymenolepis diminuta

Extracts of the tapeworm, Hymenolepis diminuta, catalyse N5,10-methylene-tetrahydrofolate-dependent release of tritium from [5-3H]dUMP, indicating the presence of thymidylate synthase. The enzyme activity was found in immature, mature and gravid proglottids, as well as in immature and mature oncospheres. The reaction showed pH optimum at 7.5. Its Michaelis constants were approximately 2 and 15 microM for dUMP and (+/-), L-N5,10-methylenetetrahydrofolate, respectively. Incubation of the tapeworm extracts with 5-F-[3H]dUMP and N5,10-methylenetetrahydrofolate resulted in formation of a labelled complex, separable under conditions of SDS polyacrylamide electrophoresis (mol. wt. of approx. 34,000), corresponding to thymidylate synthase subunit. Results of gel filtration of the above complex, under nondenaturing conditions, pointed to a dimeric structure of the enzyme.     

Trapping and partial characterization of an adduct postulated to be the covalent catalytic ternary complex of thymidylate synthase

The proposed mechanism of action of thymidylate synthase envisages the formation of a covalent ternary complex of the enzyme with the substrate dUMP and the cofactor 5,10-methylenetetrahydrofolate (CH2H4folate). The proposed structure of this adduct has been based by analogy on that of the covalent inhibitory ternary complex thymidylate synthase-FdUMP-CH2H4folate. Our recent success in using the protein precipitant trichloroacetic acid to trap the latter complex and covalent binary complexes of the enzyme with FdUMP, dUMP, and dTMP led to the use of this technique in attempts to trap the transient putative covalent catalytic ternary complex. Experiments performed with [2-14C]dUMP and [3',5',7,9-3H]CH2H4folate show that both the substrate and the cofactor remained bound to the protein after precipitation with trichloroacetic acid. The trapped putative covalent catalytic complex was subjected to CNBr fragmentation, and the resulting peptides were fractionated by reverse-phase high-pressure liquid chromatography. The isolated active site peptide was shown to retain the two ligands and was further characterized by a limited sequence analysis using the dansyl Edman procedure. The inhibitory ternary complex, which was formed with [14C]FdUMP and [3H]CH2H4folate, served as a control. The active site peptide isolated from the CNBr-treated inhibitory ternary complex was also subjected to sequence analysis. The two peptides exhibited identical sequences for the first four residues from the N-terminus, Ala-Leu-Pro-Pro, and the fifth amino acid residue was found to be associated with the labeled nucleotides and the cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Strategies for the measurement of the inhibitory effects of thymidine analogs on the activity of thymidylate synthase in intact murine leukemia L1210 cells

Two strategies have been pursued to monitor the inhibition of thymidylate (dTMP) synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) by thymidine (dThd) analogs in intact murine leukemia L1210 cells. The first method was based on the determination of tritium release from 2'-deoxy[5-3H]uridine [( 5-3H]dUrd) or 2'-deoxy[5-3H]cytidine [( 5-3H]dCyd); the second method was based on an estimation of the amount of dCyd incorporated into DNA as dTMP. The validity of these procedures was assessed by evaluating the inhibition of thymidylate synthase in murine leukemia L1210 cells by a series of 18 dThd analogs. There was a strong correlation between the inhibitory effects of the dThd analogs on the proliferation of L1210 cells on the one hand, and (i) their inhibitory effects on tritium release from [5-3H]dCyd (r = 0.926) and (ii) their inhibitory effects on the incorporation of dCyd into DNA dTMP (r = 0.921), on the other hand. Evaluation of tritium release from [5-3H]dCyd proved to be the most convenient method that has been described so far to measure thymidylate synthase activity and to follow the inhibitory effects of thymidylate synthase inhibitors in intact L1210 cells, since this method is rapid and very sensitive, and since it proved superior to the evaluation of tritium release from [5-3H]dUrd because it circumvents possible interactions of the inhibitors with thymidine kinase activity.     

Effect of an Asp80Ala substitution on the binding of dUTP and dUMP to Trypanosoma cruzi dUTPase

dUTPase (deoxyuridine 5'-triphosphate nucleotide hydrolase) is an enzyme responsible for maintaining low levels of intracellular dUTP and thus prevents uracil incorporation into DNA by DNA polymerases during replication and repair processes. The thermodynamics of binding for both dUTP and dUMP (deoxyuridine 5'-monophosphate) to the D80A mutant form of Trypanosoma cruzi dUTPase have been investigated by fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry. In the presence of magnesium, approximately a 30-fold decrease in the value of the k(cat) and a 15-fold increase in the K(m) for dUTP hydrolysis was calculated while a 5-fold decrease was observed in the affinity for dUMP. In the absence of magnesium, the affinity for dUTP binding was similar for both enzymes while that for dUMP was lowered 3-fold as a consequence of the mutation. Calorimetric titrations in several buffers with different ionization heats rendered similar proton exchanges during the binding of dUMP. Thus, apparently the side chain of Asp 80 does not seem to vary its protonation state during the binding process. The enthalpy change values for the D80A mutant hardly change with temperature and, in addition, were Mg(2+) independent. We conclude that the D80A mutation induces only a slight conformational change in the active site yet results in a significant alteration of nucleotide binding and modifies the ability of the enzyme to discriminate between dUTP and dUMP when magnesium is present.     

Thymidylate synthetase. Catalysis of dehalogenation of 5-bromo- and 5-iodo-2'-deoxyuridylate.

Tymidylate synthetase catalyzes the facile dehalogenation of 5-bromo-2'-deoxyuridylate (BrdUMP) and 5-iodo-2'-deoxyuridylate )IdUMP) to give 2'-deoxyuridylate (dUMP), the natural substrate of the enzyme. The reaction does not require folate cofactors and stoichiometrically consumes 2 equiv of thiol. In addition to dUMP, a minor product is formed during the debromination of BrdUMP which has been identified as a 5-alkylthio derivative formed by displacement of bromide ion by thiolate. The reaction has been found to proceed with a substantial alpha-secondary inverse tritium isotope effect (kT/kH = 1.212--1.258) with [2-14C,6-3H]-BrdUMP as the substrate. Similarly, an inverse tritiumisotope effect of 1.18 was observed in the nonenzymatic chemical counterpart of this reaction, the cysteine-promoted dehalogenation of [2-14C,6-3H]-5-bromo-2'-deoxyuridine. Previous evidence for the mechanism of action of this enzyme has rested largely on chemical model studies and on information obtained from its stoichiometric interaction with the inhibitor 5-fluoro-2'-deoxyuridylate. The magnitude of the secondary isotope effect during the enzymatic dehalogenation described here provides direct proof for nucleophilic catalysis and formation of 5,6-dihydroprimidine intermediates in a reaction catalyzed by thymidylate synthetase.     

Thymidylate synthetase and 2'-deoxyuridylate form a tight complex in the presence of pteroyltriglutamate.

Thymidylate synthetases of human and bacterial origin form a tightly bound complex with the substrate dUMP in the presence of pteroyltriglutamate. This complex and the weaker enzyme . dUMP binary complex can be isolated and conveniently assayed by nitrocellulose disc filtration using [6-3H]dUMP as the radioactive ligand. Intact thymidylate synthetase . dUMP . pteroyltriglutamate complex can be obtained by gel filtration chromatography on Sephadex G-25, but the binary enzyme . dUMP complex dissociates under the same conditions. Scatchard plots show the presence of two nonequivalent dUMP binding sites on the enzyme for the pteroyltriglutamate complex, with dissociation constants of 5 and 95 nM compared to 730 nM for the binary complex. The implications of these findings for folate analog inhibition of thymidylate synthetase are discussed.     

Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA

A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N3dUTP is the nitration of dUMP in 98% yield in 5 min at 25 degrees C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH2dUMP). Diazotization of 5-NH2dUMP with HNO2 followed by the addition of NaN3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N3dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N3dUTP through a chemical coupling of pyrophosphate to 5-N3dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP. When UMP was used as the starting compound, 5-N3UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare [gamma-32P]-5-N3dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing gamma-32P-labeled pyrimidine nucleotides was developed. [gamma-32P]-5-N3dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of thymidylate synthetase with 5-nitro-2'-deoxyuridylate

5-Nitro-2'-deoxyuridylate (NO2dUMP) is a potent mechanism-based inhibitor of dTMP synthetase. After formation of a reversible enzymeìnhibitor complex, there is a rapid first order loss of enzyme activity which can be protected against by the nucleotide substrate dUMP. From studies of model chemical counterparts and the NO2dUMPdTMP synthetase complex, it has been demonstrated that a covalent bond is formed between a nucleophile of the enzyme and carbon 6 of NO2dUMP. The covalent NO2dUMPènzyme complex is sufficiently stable to permit isolation on nitrocellulose membranes, and dissociates to give unchanged NO2-dUMP with a first order rate constant of 8.9 x 10(-3) min-1. Dissociation of the complex formed with [6-3H]NO2dUMP shows a large alpha-secondary isotope effect of 19%, verifying that within the covalent complex, carbon 6 of the heterocycle is sp3-hybridized. The spectral changes which accompany formation of the NO2dUMPènzyme complex support the structural assignment and, when used to tritrate the binding sites, demonstrate that 2 mol of NO2dUMP are bound/mol of dimeric enzyme. The interaction of NO2dUMP with dTMP synthetase is quite different than that of other mechanism-based inhibitors such as 5-fluoro-2'-deoxyuridylate in that it neither requires nor is facilitated by the concomitant interaction of the folate cofactor, 5,10-CH2-H4folate, and that the covalent complex formed is unstable to protein denaturants.     

Thymidylate synthetase catalyzed exchange of tritiumfrom [5-3H]-2'-deoxyuridylate for protons of water.

Thymidylate synthetase catalyzes an exchange of tritium of [5-3H]dUMP for protons of water in the absence of CH2-H4folate. The turnover number for this reaction is some 45,000-fold lower than that of dTMP formation and Km is 1.2 X 10(-5) M, similar to the dissociation constant of the enzyme-dUMP complex determined by equilibrium dialysis. The presence of 4 mM folate has no effect on Vmax but results in a decrease in the Km of dUMP to a value close to that in the normal enzymic reaction. The exchange reaction provides definitive evidence that the enzymic reaction involves attack of a nucleophile of the enzyme on the 6 position of dUMP to provide a 5,6-dihydro-dUMP intermediate which is covalently bound to the enzyme. Stereochemical considerations of the exchange reaction require proposal of a partial reaction which is not completely sterospecific or a complex reaction in which protons of water are handled with complete stereospecificity in a fashion similar to the one carbon unit of the normal enzymic reaction.     

The crystal structure of the Leishmania major deoxyuridine triphosphate nucleotidohydrolase in complex with nucleotide analogues, dUMP, and deoxyuridine

Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead anti-trypanosomal compounds.