Thymidylate synthetase from Escherichia coli K12. Purification, and dependence of kinetic properties on sugar conformation and size of the 2' substituent.

Thymidylate synthetase from Escherichia coli K12 has been purified 3600-fold by a series of chromatographic procedures. The final preparation had a specific activity of 1.47 units/mg protein and was approximately 80% pure. The enzyme is a dimer of relative molecular mass, Mr, 64000 composed of two subunits of Mr 32,000 each. Its isoelectric point is 4.7 and it is stimulated by Mg2+. Michaelis constants for (+)5,10-methylene-5,6,7,8-tetrahydrofolate [(+)CH2H4folate] were 0.014 mM in the case of methylation of 2'-deoxyuridine-5'-phosphate (dUMP) and 0.55 mM when it served as methyl-group donor for 2'-fluoro-2'-deoxyuridine-5'-phosphate (dUflMP); the corresponding Km values for dUMP and dUflMP were 0.01 mM and 0.11 mM, respectively. The activation energies for the two reactions were found to be 72.8 kJ/mol (methylation of dUMP) and 66.1 kJ/mol (methylation of dUflMP). The data support a recognition mechanism between thymidylate synthetase and that fraction of the nucleotide the sugar moiety of which is in the 2'-endo-3'-exo conformation.     

Interaction of the 5'-phosphates of the anti-HIV agents, 3'-azido-3'-deoxythymidine and 3'-azido-2',3'-dideoxyuridine, with thymidylate synthase

A study has been made of the interaction of 3'-azido-3'-deoxythymidine 5'-phosphate (AZTMP) and 3'-azido-2',3'-dideoxy-uridine 5'-phosphate (AZdUMP) with thymidylate synthase. With the enzyme from L1210 cells and the tapeworm Hymenolepis diminuta, AZTMP was a weak inhibitor competitive with respect to dUMP (Ki = 6.3 mM and 0.5 mM); hence cytotoxicity of AZT, in cells in which accumulation of AZTMP is not high, is not due to inhibition of cellular thymidylate synthase. AZdUMP, with the L1210 enzyme, was a weak substrate (competition with dUMP described by apparent Ki = 4.7 mM), excluding conversion of AZdUMP to AZTMP as a source of toxicity of 3'-azido-2',3'-dideoxyuridine. An efficient procedure is described for enzymatic phosphorylation on a preparative scale of dideoxynucleosides.     

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.     

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.     

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.     

Early Biochemical Events Occurring After Infection of Bacillus cereus 569-SP1 with Bacteriophage GSW

After infection of Bacillus cereus 569-SP1 with the 5-hydroxymethyluracil-containing phage GSW, new dTTPase, dUTPase, and dUMP-hydroxymethylase activities appear. No significant changes in activities of other pyrimidine ribonucleoside or 2'-deoxyribonucleoside triphosphate nucleotidohydrolases were detected. dUTP and dUMP inhibit the dTTPase activity, whereas dTTP failed to inhibit dUTPase activity. The K(m) value for the substrate dUTP is 10(-4) M and for dTTP is 4.85 x 10(-4) M. Thymidylate synthetase activity is inhibited only when cells are infected during the late lag or very early log phases of growth; when cells are infected with phage during mid-log, thymidylate synthetase activity is unaffected. The data support the suggestion that, although phage GSW may inhibit an otherwise expected increase in activity of thymidylate synthetase, it fails to affect the already existing activity. The data presented do not allow discrimination as to whether the phage specifies inhibition of de novo synthesis of thymidylate synthetase or the increase in activity of already existing but not fully expressed enzyme.     

Mutation of asparagine 229 to aspartate in thymidylate synthase converts the enzyme to a deoxycytidylate methylase.

The conserved Asn 229 of thymidylate synthase (TS) forms a cyclic hydrogen bond network with the 3-NH and 4-O of the nucleotide substrate dUMP. The Asn 229 to Asp mutant of Lactobacillus casei thymidylate synthase (TS N229D) has been prepared, purified, and investigated. Steady-state kinetic parameters of TS N229D show 3.5- and 10-fold increases in the Km values of CH2H4folate and dUMP, respectively, and a 1000-fold decrease in kcat. Most important, the Asp 229 mutation changes the substrate specificity of TS to an enzyme which recognizes and methylates dCMP in preference to dUMP. With TS N229D the Km for dCMP is bout 3-fold higher than for dUMP, and the Km for CH2H4folate is increased about 5-fold; however, the kcat for dCMP methylation is 120-fold higher than that for dUMP methylation. Specificity for dCMP versus dUMP, as measured by kcat/Km, changes from negligible with wild-type TS to about a 40-fold increase with TS N229D. TS N229D reacts with CH2H4folate and FdUMP or FdCMP to form ternary complexes which are analogous to the TS-FdUMP-CH2H4folate complex. From what is known of the mechanism and structure of TS, the dramatic change in substrate specificity of TS N229D is proposed to involve a hydrogen bond network between Asp 229 and the 3-N and 4-NH2 of the cytosine heterocycle, causing protonation of the 3-N and stabilization of a reactive imino tautomer. A similar mechanism is proposed for related enzymes which catalyze one-carbon transfers to cytosine heterocycles.     

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.     

Characteristics of NADPH-dependent thymidylate synthetase purified from Streptomyces aureofaciens.

NADPH-dependent thymidylate synthetase from Streptomyces aureofaciens has been purified to homogenity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on methotrexate-Sepharose 4B. The enzyme was purified 1025-fold with a 34% yield. Basic characteristics of the enzyme were determined: molecular weight of the enzyme subunit (28,000), pH and temperature optimum, effect of cations, dependency on reducing agents, Km values for dUMP, mTHF, and NADPH (3.78, 21.1, and 38.9 microM, respectively), and inhibition effect of 5-FdUMP. Binding studies revealed the enzyme mechanism to be ordered sequential: dUMP bound before mTHF. S. aureofaciens thymidylate synthetase exhibits an absolute requirement for NADPH for the enzyme activity--a unique feature not displayed by any of the thymidylate synthetases isolated so far. NADPH is not consumed during enzyme reaction, indicating its regulatory role. The properties of S. aureofaciens thymidylate synthetase show that it is a monofunctional bacterial enzyme.     

Site-directed mutagenesis of arginine 179 of thymidylate synthase. A nonessential substrate-binding residue

X-ray structural studies have shown that Arg-179 of thymidylate synthase is complexed to bound inorganic phosphate or to the 5'-phosphate of the bound substrate dUMP. The importance of Arg-179 to the structure/function of thymidylate synthase is also indicated by its complete conservation among the 17 thymidylate synthases thus far sequenced. In the present work, Arg-179 has been replaced by Thr, Ala, Lys, and Glu using site-directed mutagenesis with a mixture of four synthetic oligonucleotides as primers. The mutant proteins complement thymidylate synthase-deficient Escherichia coli and show high enzyme activity. Each of these mutants has been purified to homogeneity, partially sequenced to verify the mutation, and has had its steady state kinetic parameters determined. The most significant effect of all mutations is localized to a decrease in the net rate of association of thymidylate synthase with dUMP; the Lys mutant also shows an apparent increase in the dissociation constant of the folate cofactor of the reaction. The high activity in the mutant enzymes is explained by "plasticity" of the enzyme and compensatory actions of the other Arg residues. Why the Arg-179 residue has been conserved during evolution remains an open question.     

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.     

Different Activities of 5-Hydroxy-dUMP and 5-Hydroxymethyl-dUMP in Thymidylate Synthase-Catalyzed Reaction in View of Molecular Modeling and Structural Studies

In order to explain different activities shown by 5-hydroxy-dUMP (substrate) and its close analogue 5-hydroxymethyl-dUMP (slow-binding inhibitor) in the reaction catalyzed by thymidylate synthase, studies have been undertaken involving (i) ab initio RHF simulations, (ii) comparative analysis of crystallographic structures available from CSD, and (iii) QSAR analysis of experimental results describing thymidylate synthase interaction with various 5-substituted dUMP analogues. Assuming substrate activity of 5-hydroxy-dUMP to be associated with proton release from the C(5) hydroxyl in the enzyme-catalyzed reaction, acidities of 5-hydroxy and 5-hydroxymethyl substituents in dUMP molecule were compared. The results indicate the 5-hydroxyl deprotonation to be easier and supported by resonance electronic effect, pointing to a probable mechanism of different activities of the two dUMP analogues in thymidylate synthase reaction. The possibility is discussed that 5-mercapto-dUMP and 5-hydroseleno-dUMP, previously assumed to be inhibitors, could be also substrates for thymidylate synthase, as the 5-mercaptyl and 5-hydroselenidyl appear to be deprotonated even more easily than the 5-hydroxyl. Copyright 2000 Academic Press.     

Assay of intracellular thymidylate synthetase activity and inhibition by 5-fluoro-2'-deoxyuridine in lymphocytes.

Thymidylate synthetase catalyses the formation of thymidine monophosphate from deoxyuridine monophosphate. Purified thymidylate synthetase can be assayed radiochemically using labelled deoxyuridine monophosphate as substrate, but cells are impervious to deoxyuridine monophosphate and so intracellular thymidylate synthetase activity cannot be assayed in this way. In this paper we describe the assay of intracellular thymidylate synthetase activity in intact cells using labelled 2'-deoxyuridine. The assay showed linear kinetics with respect to time, concentration of 2'-deoxyuridine, and cell concentration. 5-fluoro-2'-deoxyuridine inhibited intracellular thymidylate synthetase activity measured with this assay by 50% at 5 nM. Cell growth was inhibited by 50% at 6 nM 5-fluoro-2'-deoxyuridine. The assay was specific for thymidylate synthetase and enabled measurement of thymidylate synthetase activity in situ in intact cells.     

Synthesis and Evaluation of 6‐Aza‐2′‐deoxyuridine Monophosphate Analogs as Inhibitors of Thymidylate Synthases,and as Substrates or Inhibitors of Thymidine Monophosphate Kinase in Mycobacterium tuberculosis

A series of 5‐substituted analogs of 6‐aza‐2′‐deoxyuridine 5′‐monophosphate, 6‐aza‐dUMP, has been synthesized and evaluated as potential inhibitors of the two mycobacterial thymidylate synthases (i.e., a flavin‐dependent thymidylate synthase, ThyX, and a classical thymidylate synthase, ThyA). Replacement of C(6) of the natural substrate dUMP by a N‐atom in 6‐aza‐dUMP 1a led to a derivative with weak ThyX inhibitory activity (33% inhibition at 50 μM ). Introduction of alkyl and aryl groups at C(5) of 1a resulted in complete loss of inhibitory activity, whereas the attachment of a 3‐(octanamido)prop‐1‐ynyl side chain in derivative 3 retained the weak level of mycobacterial ThyX inhibition (40% inhibition at 50 μM ). None of the synthesized derivatives displayed any significant inhibitory activity against mycobacterial ThyA. The compounds have also been evaluated as potential inhibitors of mycobacterial thymidine monophosphate kinase (TMPKmt). None of the derivatives showed any significant TMPKmt inhibition. However, replacement of C(6) of the natural substrate (dTMP) by a N‐atom furnished 6‐aza‐dTMP ( 1b ), which still was recognized as a substrate by TMPKmt.     

Purification and characterization of thymidylate synthetase from rat regenerating liver

Thymidylate synthetase (EC 2.1.1.45) from rat regenerating liver has been purified over 5000-fold to apparent homogeneity by a procedure involving two affinity methods. Molecular weight of the native enzyme was found to be about 68,000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band of molecular weight of 35,000, suggesting that thymidylate synthetase is a dimer of very similar or identical subunits. The Michaelis constants for deoxyuridylate (dUMP) and (+/-)L-5,10-methylenetetrahydrofolate are 6.8 microM and 65 microM, respectively. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be ordered sequential. 5-Fluoro-dUMP, halogenated analog of the nucleotide substrate is a competitive inhibitor of the enzyme, with an apparent Ki value of 5 nM. Amethopterin, analog of the cofactor is also a competitive inhibitor with an apparent Ki value of 23 microM.     

Essential arginyl residues in thymidylate synthetase.

Thymidylate synthetase from amethopterin-resistant $\text{Lactobacillus}$$\text{casei}$ is rapidly and completely inactivated by 2,3-butanedione in borate buffer, a reagent that is highly selective for the modification of arginyl residues. The reversible inactivation follows pseudo-first order kinetics and is enhanced by borate buffer. dUMP and dTMP afford significant protection against inactivation while (±)-5,10-methylenetetrahydrofolate and 7,8-dihydrofolate provide little protection. Unlike native enzyme, butanedione-modified thymidylate synthetase is incapable of interacting with 5-fluoro-2′-deoxyuridylate and 5,10-(+)-methylenetetrahydrofolate to form stable ternary complex. The results suggest that arginyl residues participate in the functional binding of dUMP.     

Protective effect of the pteroylpolyglutamates and phosphate on the proteolytic inactivation of thymidylate synthetase

Thymidylate synthetase is readily inactivated by trypsin, chymotrypsin, and carboxypeptidase A when incubated in 10–20 mm potassium phosphate buffer (pH 7.0). The loss is activity produced by trypsin and chymotrypsin is accomplished by extensive protein degradation, while inactivation by carboxypeptidase A is accompanied by release of the carboxyl-terminal valine only (Aull et al., 1974, J. Biol. Chem., 249, 1167–1172). In contrast, when the incubations are conducted in 100–200 mm potassium phosphate buffer (pH 7.0), the synthetase is not inactivated by any of the three enzymes and the results of amino acid analysis and sodium dodecyl sulfate disc gel electrophoresis demonstrate that proteolysis is prevented. The resistance of thymidylate synthetase to inactivation was shown not to be due to the inhibition of the proteolytic enzymes by the buffer. The inactivation is not prevented either by pteroylmonoglutamates or by 2′-deoxyuridine 5′-phosphate (dUMP) alone, but the presence of both is partially protective. The pteroylpolyglutamates, however, offer limited protection against carboxypeptidase A and chymotrypsin; in combination with dUMP, proteolytic inactivation of the snythetase by all three enzymes is prevented. Characterization of the properties of carboxypeptidase A-inactivated thymidylate synthetase reveals the following, (i) The binding of deoxynucleotides is unaltered, but the binding of a variety of pteroylpolyglutamate derivatives is reduced or abolished, (ii) Pteroylpolyglutamates are bound provided dUMP or an analog such as 5-fluorodUMP is present, (iii) Ternary complex formation between carboxypeptidase A-inactivated enzyme and (+)5,10-methylenetetrahydropteroyltetraglutamate plus 5-fluorodUMP occurs in the same molar binding ratio (1:2:2) at saturation as with the native enzyme, but differs from the native enzyme ternary complex in that the dissociation constant for 5-fluorodUMP is increased by approximately 10⁵. In addition, there is no evidence for the formation of covalent linkages between the ligands and enzyme, (iv) The treated enzyme cannot catalyze tritium release from [³H₅]dUMP in the presence of either (+)5,10-methylenepteroylmonoglutamate or (+)5,10-methylenetetrahydropteroyltetraglutamate.     

The role of protein dynamics in thymidylate synthase catalysis: variants of conserved 2'-deoxyuridine 5'-monophosphate (dUMP)-binding Tyr-261

The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate. Using kinetic and X-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3'-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Km values for both the substrate and cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from the bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of Escherichia coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.     

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.     

Kinetic properties of human thymidylate synthase,an anticancer drug target

We have determined the kinetic parameters of human recombinant thymidylate synthase (hrTS) with its natural substrate, dUMP, and E-5-(2-bromovinyl)-2(')-deoxyuridine monophosphate (BVdUMP), a nucleotide derivative believed to be the active species of the novel anticancer drug NB1011. NB1011 is activated by hrTS and is selectively toxic to high thymidylate synthase expressing tumor cells. BVdUMP undergoes hrTS-catalyzed thiol-dependent transformation. dUMP and BVdUMP act as competitive hrTS substrates. The natural folate cofactor, CH(2)-THF, inhibits the TS-catalyzed reaction with BVdUMP. We suggest that lower folate levels found in tumor cells favor TS-catalyzed BVdUMP transformation, which, in addition to higher levels of TS expression in tumor cells, contributes to the favorable therapeutic index of the drug NB1011.