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.     

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.     

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.     

The effect of Arg209 to Lys mutation in mouse thymidylate synthase

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.     

Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei

Pattanakitsakul S. and Ruenwongsa P. 1984. Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei. International Journal for Parasitology14: 513–520. Thymidylate synthetase (TS) and dihydrofolate reductase (DHFR) from Plasmodium berghei were copurified by Sephacryl S-300 and Sephadex G-200 column chromatography and found to have an apparent mol. wt of 132,000. Electrophoresis of the partially purified enzyme under non-denaturing conditions showed the comigration of TS and DHFR. The mol. wt of TS was estimated to be 65,000 on SDS-gel electrophoresis. Both enzymes exhibit a broad pH optimum in the range of 6.5–8.0. Urea, NaCl and KC1 inhibit TS but activate DHFR. For TS, the apparent K_m for dUMP and methylene-tetrahydrofolate have been found to be 71.4 and 312.5 μM, respectively. For DHFR, the apparent K_m for dihydrofolate and NADPH have been found to be 4.4 and 12.5 μM, respectively. Inhibition of DHFR by pyrimethamine, methotrexate and trimethoprim are competitive with dihydrofolate with K_is of 0.63, 0.5 and 1.88 nM, respectively. FdUMP inhibition of TS is competitive with dUMP with K_is of 0.05 μM, but inhibition by methotrexate is uncompetitive with dUMP and MTHF with K_ii of 103 and 23 μM, respectively.     

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.     

5'-Haloacetamido-5'-deoxythymidines: novel inhibitors of thymidylate synthase

5'-Bromoacetamido-5'-deoxythymidine (BAT), 5'-iodoacetamido-5'-deoxythymidine (IAT), 5'-chloroacetamido-5'-deoxythymidine (CAT) and [14C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were investigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT greater than IAT greater than CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4-8-fold higher than it was in the absence of dUMP. The I50 values for BAT were 1 X 10(-5) M and 1.2 X 10(-6) M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thymidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The Km for the enzyme was 9.2 microM, and the Ki determined for competitive inhibition by BAT was 5.4 microM. Formation of a tight, irreversible complex is inferred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min-1, and the steady-state constant of inactivation, Ki, was estimated to be 6.6 microM. The 5'-haloacetamido-5'-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.     

2''-Deoxy-2''-fluorouridine-5''-phosphate: an alternative substrate for thymidylate synthetase from Escherichia coli K12.

The substrate specificity of 2'-deoxy-2'-substituted uridines and their 5'-phosphates towards thymidylate synthetase from Escherichia coli K12 was investigated. Besides the natural substrate 2'-deoxyuridine-5'-phosphate (dUMP), only 2'-deoxy-2'-fluorouridine-5'-phosphate (dUflMP) was a substrate. The KM of dUflMP is 11 times higher than that of dUMP, while the Vmax values are virtually the same. It is concluded that the size of the 2'-substituent and not its polarity (and the concomitant conformational change) determines substrate specificity of thymidylate synthetase.     

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.     

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.     

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.     

Demonstration of separate binding sites for the folate coenzymes and deoxynucleotides with inactivated Lactobacilluscasei thymidylate synthetase

In contrast to (+)5,10-methylenetetrahydropteroylmonoglutamate which does not bind to $\text{Lactobacillus}\text{casei}$ thymidylate synthetase, the corresponding tetraglutamate analog binds to a single site with a K_D = 2 × 10^?5 M. Alkylation of one of the enzyme's four cysteines with N-ethylmaleimide or iodoacetate prevented the binding of dUMP, but did not affect the binding of the pteroyltetraglutamate. Inactivation of the synthetase with carboxypeptidase A, however, prevented the binding of (+)5,10-methylenetetrahydropteroyltetraglutamate but not that of dUMP. The binding of (+)5,10-methylenetetrahydropteroyltetraglutamate to native enzyme was associated with the appearance of a positive circular dichroic band at 303 nm ([θ] = 7 × 10⁴ deg·cm²dmol^?1). The latter effect was not impaired by the inhibition of the enzyme with N-ethylmaleimide, whereas formation of the ternary complex, coenzyme-synthetase-FdUMP, was prevented by alkylation. These studies reveal that thymidylate synthetase can be inactivated in a manner that does not prevent the binding of the substrates individually.     

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 tyrosyl residues in Lactobacillus casei thymidylate synthetase

Sulfhydryl-blocked thymidylate synthetase (EC 2.1.1.4.5) is rapidly inactivated by low concentrations of tetranitromethane. This reagent first nitrates two non-essential tyrosines per dimeric enzyme molecule followeed by two essential tyrosines with no oxidation of sulfhydryl groups. dUMP affords significant protection against inactivation. These results suggest that essential tyrosyl residues are present in the active sites of the enzyme.     

Kinetic behaviour and allosteric regulation of human deoxycytidylate deaminase derived from leukemic cells

Deoxycytidylate deaminase has been highly purified (1232-fold) from human leukemia CCRF-CEM cells. The native molecular weight of the enzyme is 108 000 and subunit molecular weight 50 500, suggesting that the native enzyme exists as a dimer. The enzyme exhibits a sigmoidal initial velocity vs substrate concentration curve and is regulated by allosteric effectors, dCTP and TTP. The curve relating substrate concentration to initial velocity was changed from a sigmoidal shape to a hyperbolic one by the activator dCTP, while the inhibitor TTP increased the sigmoidicity of the curve. The molecular weight of deoxycytidylate deaminase was unchanged in the presence of allosteric effectors, indicating that aggregation-disaggregation is not the basis of regulation. Deoxycytidylate deaminase exhibited the greatest affinity for the substrate dCMP, with lesser affinity for ara-CMP, and least affinity for CMP. Ara-CMP was an effective substrate in the presence of dCTP concentrations exceeding 4 microM. These data indicate that human neoplastic cell deoxycytidylate deaminase is a highly regulated allosteric enzyme, which is likely to have a significant influence on cellular dUMP, dCTP and TTP pools. These findings further suggest, that the enzyme through its influence on dUMP levels is likely to modulate the biochemical effects of pyrimidine antimetabolites active against the thymidylate synthetase reaction and in the presence of elevated dCTP pools will promote deamination of ara-CMP to the inactive ara-UMP.     

Using logic programming for modeling the one-carbon metabolism network to study the impact of folate deficiency on methylation processes

Dynamical modeling is an accurate tool for describing the dynamic regulation of one-carbon metabolism (1CM) with emphasis on the alteration of DNA methylation and/or dUMP methylation into dTMP. Using logic programming we present a comprehensive and adaptative mathematical model to study the impact of folate deficiency, including folate transport and enzymes activities. 5-Methyltetrahydrofolate (5mTHF) uptake and DNA and dUMP methylation were studied by simulating nutritional 5mTHF deficiency and methylenetetrahydrofolate reductase (MTHFR) gene defects. Both conditions had distinct effects on 1CM metabolite synthesis. Simulating severe 5mTHF deficiency (25% of normal levels) modulated 11 metabolites. However, simulating a severe decrease in MTHFR activity (25% of normal activity) modulated another set of metabolites. Two oscillations of varying amplitude were observed at the steady state for DNA methylation with severe 5mTHF deficiency, and the dUMP/dTMP ratio reached a steady state after 2 h, compared to 2.5 h for 100% 5mTHF. MTHFR activity with 25% of V(max) resulted in an increased methylated DNA pool after half an hour. We observed a deviation earlier in the profile compared to 50% and 100% V(max). For dUMP methylation, the highest level was observed with 25%, suggesting a low rate of dUMP methylation into dTMP with 25% of MTHFR activity. In conclusion, using logic programming we were able to construct the 1CM for analyzing the dynamic system behavior. This model may be used to refine biological interpretations of data or as a tool that can provide new hypotheses for pathogenesis.     

Purification of mammalian tumor (L1210) thymidylate synthetase by affinity chromatography on stable biospecific adsorbent. Stabilization of the enzyme with neutral detergents.

Thymidylate synthetase from mouse leukemic L1210 cells was purified to electrophoretic homogeneity with 70% yield as a result of an affinity chromatography procedure based on reversible deoxyuridylate-dependent binding of the enzyme to a stable biospecific adsorbent, 10-formyl-5,8-dideazafolate, immobilized on aminoethyl-Sepharose. The presence of neutral detergents, Triton X-100, or Nonidet P40 stabilized thymidylate synthetase during purification. Analytical electrophoresis of the enzyme treated with an excess of 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate showed the presence of two forms of thymidylate synthetase--5-fluorodeoxyuridylate.5,10-methylenetetrahydrofolate complex, indicating that there are two binding sites for 5-fluorodeoxyuridylate present on the enzyme molecule. Molecular weight of native thymidylate synthetase was found to be 75,000, whereas that for the monomer was 38,500.     

Human thymidylate synthetase derived from blast cells of patients with acture myelocytic leukemia. Purification and chracterization.

Thymidylate synthetase (EC 2.1.1.B.) from blast cells of patients with acute myelocytic leukemia has been purified more than 1470-fold by affinity column chromatography. Methotrexate was the affinity ligand. dUMP was found to be a necessary additive for retention of the enzyme by the affinity column. Disc electrophoresis and sucrose density gradient centrifugation revealed a single enzyme species with a molecular weight of 76,000. The enzyme exhibits a temperature-dependent conformational change with activation energies of 5.3 +/- 0.4 and 17.3 +/- 1.9 kcal/mol, respectively, above and below a transitional temperature of 35 degrees. This conformational change is reflected in the binding affinity of dUMP but not of 5,10-methylenetetrahydrofolate. The enzyme displays a broad pH maximum in the range of pH 7.4 to 8.8. The Michaelis constants for dUMP and (+/--L-5,10-methylenetetrahydrofolate are 1.8 +/- 0.2 and 31 +/- 8.3 micrometer, respectively. Initial velocity and product inhibition studies reveal the enzymic mechanism to be ordered sequential. dUMP binds before 5,10-methylenetetrahydrofolate and dihydrofolate is released before TMP. 5-Fluoro-2'-deoxy-5'-uridylate (FdUMP) behaves as in irreversible inhibitor with a Ki of 1.68 +/- 0.45 nM. The enzyme has a turnover number of 6 min-1 per FdUMP binding site. Methotrexate inhibits noncompetitively with respect to dUMP and binds tighter to the enzyme in the presence of dUMP. Methotrexate antagonizes inactivation of the enzyme by FdUMP.     

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.     

Alteration in properties of thymidylate synthetase from pyrimethamine-resistant Plasmodium chabaudi

Alteration in properties of thymidylate synthetase from pyrimethamine-resistant smodium chabaudi. International Journal for Parasitology16: 483–490. Thymidylate synthetase from cloned strains of pyrimethamine-sensitive and resistant P. chubaudi were partially purified and characterized. The enzyme from both strains have equal mol. wt of 120,000 as estimated by Sephadex G-200 column chromatography. The enzyme from drug-sensitive parasites has an optimum pH of 6.5–7.5 and is stable at pH 4–11 while that from drug-resistant strain has an pH optimum of 7.0–8.0 and is stable at pH 5–10. The K_m for methylenetetrahydrofolate are 206 ± 6 and 495 ± 5 μm for the enzyme from drug-resistant and sensitive parasites, respectively. The K_m for dUMP of the enzyme from drug-resistant and sensitive parasites are 42 ± 1 and 49 ± 6 μm, respectively. Inhibition of the enzyme from both strains by FdUMP are competitive with dUMP; however,the K_is for the enzyme from drug-resistant strain (0.043 ± 0.005 μm) is less than that from drug-sensitive strain (0.11 ± 0.007 μm) by a factor of 2.5. The K_ii for methotrexate with respect to methylenetetrahydrofolate of the enzyme from drug-resistant parasites (58 ± 3 μm) is 3 times larger than that from drug-sensitive parasites (17 ± 1 μm).