Evidence for the existence of covalent nucleotide-thymidylate synthase complexes, identification of site of attachment, and enhancement by folates

The formation of covalent binary complexes of thymidylate synthase and its nucleotide substrate dUMP, product dTMP, and inhibitor, 5-fluorodeoxyuridylate (FdUMP) was investigated using the trichloroacetic acid precipitation method. It was observed that, in addition to FdUMP, both dUMP and dTMP were capable of covalent interactions with the enzyme in the absence of added folates. The presence of folate, dihydrofolate, or tetrahydrofolate (H4folate) was found to produce substantial enhancements in the covalent binding of both FdUMP and dUMP to the enzyme with H4folate being the most effective agent. Further, covalent binary complexes of the enzyme with the three radiolabeled nucleotides were isolated by trichloroacetic acid precipitation and subjected to CNBr cleavage. The active-site CNBr peptide was isolated by reverse phase high performance liquid chromatography, and the first five N-terminal amino acid residues were sequenced by the dansyl-Edman procedure. Each active site peptide obtained from the covalent binary complexes as well as that from the covalent inhibitory ternary complex formed from enzyme, FdUMP, and 5,10-methylene-H4folate exhibited an identical sequence of Ala-Leu-Pro-Pro-(X)-, and the 5th amino acid was found to be associated with radiolabeled nucleotide ligand. Dansyl-Edman sequence analysis of the active site CNBr peptide, derived from enzyme which had been treated with iodoacetic acid, gave a sequence of Ala-Leu-Pro-Pro-CmCys (where CmCys is carboxymethylcysteine), thus confirming the fact that the fifth residue from the N terminus is Cys-198. In all the cases, the active site Cys-198 residue was found to be covalently linked to the nucleotides. These results provide unequivocal proof that the covalent binary complexes of enzyme with dUMP and dTMP predicted in the catalytic reaction mechanism actually exist.     

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)     

Thymidylate synthase with a C-terminal deletion catalyzes partial reactions but is unable to catalyze thymidylate formation.

The V316Am mutant of Lactobacillus casei thymidylate synthase has a single amino acid deletion at the C-terminus which abolishes catalysis of dTMP formation. However, V316Am catalyzes two partial reactions which require covalent catalysis: a CH2H4folate-dependent exchange of the 5-hydrogen of dUMP for protons in water and a thiol-dependent dehalogenation of 5-bromo- and 5-iodo-dUMP. These reactions proceed with kcat and Km values similar to those of the wild-type TS-catalyzed reactions. dUMP, dTMP, and FdUMP are competitive inhibitors of the debromination reaction with Ki values similar to those obtained with wild-type enzyme. These results show that removal of the terminal valine does not alter the ability of the enzyme to bind to or form covalent bonds with nucleotide ligands. V316Am also forms a covalent ternary complex with FdUMP and CH2H4folate. However, the affinity of the TS-FdUMP complex for the cofactor is reduced, and the rate of covalent ternary complex formation and its stability are significantly lower than with wild-type TS. These results allow us to place the major defects of the mutation on steps that occur subsequent to initial CH2H4folate binding.     

Evidence from chemical degradation studies for a covalent bond from 5-fluoro-2'-deoxyuridylate to N-5 of tetrahydrofolate in the ternary complex of thymidylate synthetase-5-fluoro-2'-deoxyuridylate-5,10-methylenetetrahydrofolate

In the ternary complex of thymidylate synthetase, 5-fluoro-2'-deoxyuridylate (FdUMP), and 5,10-methylenetetrahydrofolate (5,10-CH2H4folate), the 5-fluorouracil moiety is covalently bound to the enzyme by a sulfide linkage from C-6 and to either N-5 or N-10 of H4folate by a methylene bridge from C-5. In an effort to establish the site by which H4folate is attached to FdUMP, the ternary complex was subjected to reagents that cleave the C-9, N-10 bond of folate derivatives. The complex was stable to zinc dust in hydrochloric acid, a reagent that cleaves N-10-substituted but not N-5-substituted folates. The conditions of the Bratton-Marshall reaction, which involve the use of nitrous acid, were found to cleave N-5-substituted folates in yields ranging from 5 to 50%. Exposure of the double-labeled thymidylate synthetase-FdUMP-[2-14C,7,9,3',5'-3H]5,10-CH2H4folate complex to the Bratton-Marshall reaction resulted in 16% cleavage of the C-9, N-10 bond with release solely of p-aminobenzoylglutamate, whereas all of the carbon-14-labeled pterin residue remained covalently bound to the protein. These results demonstrate that in the ternary complex, the 5-fluorouracil residue is connected by a covalent bond to N-5 of H4folate.     

A stable binary complex between Leishmania major thymidylate synthase and the substrate deoxyuridylate. A slow-binding interaction

The thymidylate synthase (TS) activity in Leishmania major resides on the bifunctional protein thymidylate synthase-dihydrofolate reductase (TS-DHFR). We have isolated, either by Sephadex G-25 chromatography or by nitrocellulose filter binding, a binary complex between the substrate deoxyuridylate (dUMP) and TS from L. major. The kinetics of binding support a "slow binding" mechanism in which dUMP initially binds to TS in a rapid, reversible pre-equilibrium step (Kd approximately 1 microM), followed by a slow first-order step (k = 3.5 X 10(-3) s-1) which results in the isolable complex; the rate constant for the dissociation of dUMP from this complex was 2.3 X 10(-4) s-1, and the overall dissociation constant was approximately 0.1 microM. The stoichiometry of dUMP to enzyme appears to be 1 mol of nucleotide bound/mol of dimeric TS-DHFR. Binary complexes between the stoichiometric inhibitor 5-fluorodeoxyuridylate (FdUMP) and TS, and between the product deoxythymidylate (dTMP) and TS were also isolated by nitrocellulose filter binding. Competition experiments indicated that each of these nucleotides were binding to the same site on the enzyme and that this site was the same as that occupied by the nucleotide in the FdUMP-cofactor X TS ternary complex. Thus, it appeared that the binary complexes were occupying the active site of TS. However, the preformed isolable dUMP X TS complex is neither on the catalytic path to dTMP nor did it inhibit TS activity, even though the dissociation of dUMP from this complex is several orders of magnitude slower than catalytic turnover (approximately 3 s-1). The results suggest that dUMP binds to one of the two subunits of the native protein in a catalytically incompetent form which does not inhibit activity of the other subunit.     

Properties of a defined mutant of Escherichia coli thymidylate synthase

A mutant of Escherichia coli thymidylate synthase (F3-TS), resulting from the replacement of a tyrosine for a cysteine 50 amino acids from the amino-terminal end, has been purified to homogeneity and found to contain less than 0.2% of the activity of the native enzyme (thyA-TS). Although this protein formed a ternary complex with 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and 5,10-methylenetetrahydrofolate, like the native enzyme, the extent of complex formation was significantly impaired as determined by equilibrium dialysis and circular dichroism. Thus, unlike the native enzyme, where 2 mol of FdUMP were present in each mole of ternary complex, F3-TS contained less than 1 mol of FdUMP/mol of ternary complex. Similarly, the binding of dUMP by F3-TS was greatly diminished relative to thyA-TS, but its binding as well as that of FdUMP could be improved by the presence of either the folate substrate or a tight binding folate analogue, 10-propargyl-5,8-dideazafolate (PDDF). However, despite the fact that PDDF enhanced the binding of FdUMP and dUMP to F3-TS, the binding of PDDF to the mutant enzyme was also greatly impaired. This contrasts with the native enzyme, which, under the same conditions, bound about 2 mol of PDDF/mol of enzyme in the presence or absence of either FdUMP or dUMP. Circular dichroism analyses with PDDF in the presence of dUMP or FdUMP yielded analogous results, but the effects were less dramatic than those obtained by equilibrium dialysis. Evidence in support of a structural difference between thyA-TS and F3-TS was obtained by demonstrating that the latter protein was 15-fold slower in forming a ternary complex with dUMP and PDDF than the former and that the mutant enzyme was less stable than the native enzyme.     

On the mechanism of 2'-deoxyuridylate hydroxymethylase

dUMP hydroxymethylase from SP01-infected Bacillus subtilis has been purified 160-fold by chromatography on DEAE-cellulose and ethylagarose. The enzyme catalyzes exchange of the 5-hydrogen of dUMP for protons of water in the presence or absence of the cofactor CH2-H4folate. Upon treatment with FdUMP and CH2-H4folate, an isolable covalent complex is formed which is believed to be structurally similar to a steady-state intermediate of the normal reaction. The FdUMP-CH2-H4folate-dUMP hydroxymethylase complex is stable toward denaturation with sodium dodecyl sulfate and shows a subunit molecular weight of 46 000. By analogy with chemical models and studies of dTMP synthetase, a mechanism is proposed for the reaction catalyzed by dUMP hydroxymethylase.     

Characteristics of thymidylate synthase purified from a human colon adenocarcinoma

Thymidylate synthase has been purified greater than 4000-fold from a human colon adenocarcinoma maintained as a xenograft in immune-deprived mice. In this disease, the enzyme is an important target for the cytotoxic action of 5-fluorouracil, which is influenced by the reduced folate substrate CH2-H4PteGlu. Due to the importance of this interaction, and the existence in cells of folate species as polyglutamyl forms, the interaction of folylpolyglutamates with thymidylate synthase was examined. Polyglutamates of PteGlu were used as inhibitors, and the interaction of CH2-H4PteGlu polyglutamates as substrates or in an inhibitory ternary complex were also examined. Using PteGlu1-7, Ki values were determined. A maximal 125-fold decrease in Ki was observed between PteGlu1 and PteGlu4; further addition of up to three glutamyl residues did not result in an additional decrease in Ki. Despite the increased binding affinity of folypolyglutamates for this enzyme, no change in the Km values for either dUMP (3.6 microM) or CH2-H4PteGlu (4.3 microM) were detected when polyglutamates of [6R]CH2-H4PteGlu were used as substrates. Product inhibition studies demonstrated competitive inhibition between dTMP and dUMP in the presence of CH2-H4PteGlu5. In addition, CH2-H4PteGlu4 stabilized an inhibitory ternary complex formed between FdUMP, thymidylate synthase, and CH2-H4PteGlu4. Thus the data do not support a change in the order of substrate binding and product release upon polyglutamylation of CH2-H4PteGlu reported for non-human mammalian enzyme. This is the first study to characterize kinetically thymidylate synthase from a human colon adenocarcinoma.     

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.     

A method for the determination of total,free, and 5-fluorodeoxyuridylate-bound thymidylate synthase in cell extracts

A radiochemical assay for thymidylate synthase (EC 2.1.1.45, dTMP synthase), which permits the accurate determination of total, free, and 5-fluoro-2′-deoxyuridylate (FdUMP)-bound enzyme in cells exposed to the 5-fluoropyrimidine anticancer agents, is described. The total intracellular concentrations of dTMP synthase (free plus FdUMP-bound enzyme) in extracts from CCRF-CEM leukemic cells incubated with 5-fluoro-2′-deoxyuridine were determined following dissociation of the covalent dTMP synthase-5,10-methylenetetrahydrofolate-FdUMP ternary complex in the presence of the substrate, 2′-deoxyuridine-5′-monophosphate. The addition of substrate prevented reformation of the ternary complex during the dissociation procedure, and allowed complete recovery of FdUMP binding sites in cells exposed to a high concentration of 5-fluoro-2′-deoxyuridine. After removal of the substrate by charcoal adsorption, the concentration of total FdUMP binding sites was determined by titration of the enzyme with a saturating concentration of [6-³H]FdUMP and 5,10-methylenetetrahydrofolate. The concentration of FdUMP-bound dTMP synthase was then calculated as the difference between the total and free (without prior ternary complex disruption) enzyme values. The high sensitivity of this assay coupled with its ability to accurately quantitate both free and FdUMP-bound dTMP synthase in cells exposed to a wide range of fluoropyrimidine concentrations should make it useful for a variety of experimental and clinical studies.     

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.     

Human leukemic cells resistant to 5-fluoro-2'-deoxyuridine contain a thymidylate synthetase with lower affinity for nucleotides

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.     

Catalytic mechanism of Chlamydia trachomatis flavin-dependent thymidylate synthase

Here we report on a Chlamydia trachomatis gene that complements the growth defect of a thymidylate synthase-deficient strain of Escherichia coli. The complementing gene encodes a 60.9-kDa protein that shows low level primary sequence homology to a new class of thymidylate-synthesizing enzymes, termed flavin-dependent thymidylate synthases (FDTS). Purified recombinant chlamydial FDTS (CTThyX) contains bound flavin. Results with site-directed mutants indicate that highly conserved arginine residues are required for flavin binding. Kinetic characterization indicates that CTThyX is active as a tetramer with NADPH, methylenetetrahydrofolate, and dUMP required as substrates, serving as source of reducing equivalents, methyl donor, and methyl acceptor, respectively. dTMP and H(4)folate are products of the reaction. Production of H(4)folate rather than H(2)folate, as in the classical thymidylate synthase reaction, eliminates the need for dihydrofolate reductase, explaining the trimethoprim-resistant phenotype displayed by thyA(-) E. coli-expressing CTThyX. In contrast to the extensively characterized thyA-encoded thymidylate synthases, which form a ternary complex with substrates dUMP and CH(2)H(4)folate and follow an ordered sequential mechanism, CTThyX follows a ping-pong kinetic mechanism involving a methyl enzyme intermediate. Mass spectrometry was used to localize the methyl group to a highly conserved arginine, and site-directed mutagenesis showed this arginine to be critical for thymidylate synthesizing activity. These differentiating characteristics clearly distinguish FDTS from ThyA, making this class of enzymes attractive targets for rational drug design.     

Author index

Thymidylate synthetase has been purified from cultures of Escherichia coli infected with bacteriophages T4 or T5, with the T4 enzyme being purified to at least 50% of homogeneity, and both enzymes being resolved from the corresponding host enzyme. The molecular weights are 58,000 for the T4 enzyme and 55,000 for the T5 enzyme, as estimated by gel filtration and confirmed for the T4 enzyme by sucrose gradient analysis. Disc gel electrophoresis of the T4 enzyme in sodium dodecyl sulfate gives a single band with a molecular weight of 29,000, suggesting that the enzyme is composed of two subunits. Kinetic analysis of the inhibition of the T4 enzyme by 5-fluorodeoxyuridylate (FdUMP) gives results similar to those earlier reported for the T2 and T6 enzymes. Inhibition is competitive with respect to deoxyuridylate (dUMP) if the enzyme is not preincubated with inhibitor, but a brief preincubation of enzyme and inhibitor in the presence of 5, 10-methylenetetrahydrofolate generates a pattern of noncompetitive, stoichiometric inhibition. FdUMP remains bound to the enzyme through gel filtration chromatography, consistent with various observations that this inhibitor is covalently bound. However, the enzyme-inhibitor complex is dissociated by treatment with sodium dodecyl sulfate prior to chromatography. Moreover, in contrast to studies on thymidylate synthetase from other sources, oxidation of tetrahydrofolate by FdUMP-inhibited enzyme could not be detected. Inhibition of the T5 enzyme by FdUMP is not stoichiometric, and the enzyme-inhibitor complex is readily dissociated by gel filtration. These findings suggest that there are significant differences in mechanism of FdUMP binding by thymidylate synthetases of different origins. Inhibition of the T4 enzyme by trifluoromethyldeoxyuridine 5′-monophosphate (F₃dTMP) follows the kinetics of stoichiometric inhibition, but data from both gel filtration and enzyme-inhibitor titration indicate that the enzyme binds 12–13 times as much F₃dTMP as FdUMP, suggesting that most of the F₃dTMP is bound at noncatalytic sites.     

Quinazoline folate analogs inhibit the catalytic activity of thymidylate synthase but allow binding of 5-fluorodeoxyuridylate

We have investigated some unusual aspects of the inhibition of mammalian thymidylate synthase (TS) by the folate antimetabolite, 10-propargyl-5,8-dideaza-folic acid (CB 3717). From our results, we conclude that binding of CB 3717 metabolites to one subunit of L1210 TS modified the conformation of the second active site of this enzyme so that it retained the ability to bind 5-fluro-2'-deoxyuridine-5'-monophosphate (FdUMP) but not its catalytic activity. Exposure of intact mouse L1210 cells to CB 3717 resulted in inactivation of cellular TS activity, yet desalted cytosol preparations from these cells retained the ability to bind FdUMP. The same effect was found with several analogs of CB 3717. Complexes of FdUMP formed in vitro with TS from cells exposed to CB 3717 were covalent and co-migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with complexes of FdUMP, folate cofactor, and TS from cells not exposed to CB 3717. In the presence of dUMP, a tightly bound complex rapidly formed between isolated pure TS and the pentaglutamate of CB 3717 but not the monoglutamate form of this compound. Binding experiments using CB 3717 pentaglutamate-inhibited TS suggested a stoichiometry of 1 mol of FdUMP bound per mol of dimeric TS.     

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.     

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.     

Interaction of pyridoxal phosphate with thymidylate synthase: Spectral and equilibrium dialysis studies

• 1.1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents.
• 2.2. The dissociation constant of the thymidylate synthase-PLP complex determined by equilibrium dialysis was 9 ± 1.6 μM, the maximum number of PLP molecules bound per molecule of native thymidylate synthase was 2.5 ± 0.4, and the Hill coefficient was 0.97.
• 3.3. No evidence of PLP binding was found with denatured thymidylate synthase, and only slight binding was observed when enzyme SH groups were blocked or when the active site was blocked with 5-fluorodeoxyuridylate (FdUMP) and methylenetetrahydrofoliate.
• 4.4. The presence of dUMP, dTMP, or FdUMP interfered with the binding of PLP to thymidylate synthase, and the presence of equimolar amounts of PLP interfered with the binding of dUMP.