Amino acid sequence at the FdUMP binding site of thymidylate synthetase.

Bellisario, Ronald; Maley, Gladys F.; Galivan, John; Maley, Frank

doi:10.1073/pnas.73.6.1848

Cited by 42 publications

(19 citation statements)

References 29 publications

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“…The human enzyme has considerably more residues of cysteine, glycine, and arginine and fewer of histidine than the bacterial enzyme. The 2.5-fold more cysteine residues in the human enzyme are especially interesting because a cysteine residue is catalytically active (13,31,32) and binds FdUMP (28,33) We hope that the facile purification of high-activity human thymidylate synthetase described in this report will stimulate further investigations into the mechanisms of action and inhibition of this important enzyme from human sources.…”

Section: Discussionmentioning

confidence: 99%

Thymidylate synthetase purified to homogeneity from human leukemic cells.

Lockshin¹,

Moran²,

Danenberg³

1979

Proc. Natl. Acad. Sci. U.S.A.

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Thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) from a human leukemic cell line has been purified to homogeneity with one-step affinity column chromatography. The purified enzyme has a specific activity of 3.8 jtmo /min per mg of protein, which corresponds to a turnover number of 250. These are the highest values reported for a thymidylate synthetase from neoplastic tissue. A ratio of 1.7 mol of 5-fluoro-2'.deoxyuridylate binds per mol of enzyme in the presence of 5,10-methylenetetrahydrofolate. The ternary complex so formed migrates intact on denaturing gels and can be precipitated with trichloroacetic acid; however, urea dissociates the ternary complex. The human thymidylate synthetase is composed of two subunits of 33,000 daltons each. It contains more residues of cysteine, glycine, and arginine and fewer of histidine than the well-studied thymidylate synthetase from Lactobacillus casei. Thymidylate synthetase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) has been the object of considerable interest because of its key function in mammalian DNA synthesis as the only de novo source of thymidylate. This enzyme is believed to be the major site of action of the cancer chemotherapeutic drug 5-fluorouracil due to inhibition by the metabolite 5-fluoro-2'-deoxyuridylate (FdUMP) (1). FdUMP, 5,10-methylenetetrahydrofolate (5,10-CH2H4PteGlu), and thymidylate synthetase form a ternary complex (2), which is joined by covalent bonds in some cases-i.e., is stable to protein denaturants and limited proteolysis (3)(4)(5)(6). In other cases, the ternary complex is dissociated partially (7) (16) and was used without purification. H4MTX was lyophilized in ampules, sealed in N2, and stored in the dark at -25°C.Aminoethyl polyacrylamide gel beads (Aminoethyl Bio-Gel P-150) were purchased from Bio-Rad, as were the reagents for polyacrylamide gel electrophoresis. Sephadex G-100 was obtained from Pharmacia. Other biochemicals were from Sigma.Cell Culture. CCRF-CEM human lymphoblastic leukemia cells (17) were thawed from mycoplasma-free stocks at 21/2-month intervals and were monitored routinely for absence of mycoplasma. These cells were grown in suspension in RPMI 1640 medium (GIBCO) supplemented with 10% fetal calf serum (GIBCO) and were harvested in late logarithmic growth, washed once with phosphate-buffered saline, and used immediately or stored in liquid N2.Affinity Column Preparation. Aminoethyl polyacrylamide (0.5 g) was hydrated with water and reacted with 20 mg of H4MTX and 150 mg of 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide-HCI by the method of Slavik et al. (12), except that N2 gas was used. About 40% of the resultant gel was transferred into a small plastic column (5-ml Pipetman tips partially stuffed with glass wool) in a cold room (40C). The light-tan beads were washed overnight with 200 ml of 2.5% NaHCO3/0.5 M KCl containing 0.15 M 2-mercaptoethanol. The columns were then washed with 100 ml of 10 mM potassium phosphate buffer (pH 7.4) c...

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Section: Discussionmentioning

confidence: 99%

Thymidylate synthetase purified to homogeneity from human leukemic cells.

Lockshin¹,

Moran²,

Danenberg³

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…In the course of this reaction a methylene group and hydride ion are contributed by CH2hPteGlu to the 5-position of dUMP, the latter being activated by a nucleophilic attack at the 6-position of dUMP (Pogolotti & Santi, 1977). The nucleophile was shown subsequently to be a specific cysteinyl residue of the enzyme (Bellisario et al, 1976;Pogolotti et al, 1976) and verified more recently by X-ray crystallographic studies on the 0006-2960/95/0434-1469$09.00/0 0 1995 American Chemical Society liganded structure of Escherichia coli TS (Matthews et al, 1990a,b;Montfort et al, 1990).…”

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confidence: 94%

Complete Restoration of Activity to Inactive Mutants of Escherichia coli Thymidylate Synthase: Evidence that E. coli Thymidylate Synthase is a Half-the-Sites Activity Enzyme

Maley¹,

Pedersen-Lane²,

Changchien³

1995

Biochemistry

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Escherichia coli thymidylate synthase (TS) is a dimeric protein containing identical subunits. When R126E, an inactive mutant of this enzyme, was incubated at room temperature with other inactive mutants of E. coli, TS enzyme activity gradually reappeared. The rate of activity restoration was dependent on the mutant employed. In the case of C146W, an active site mutant, the half-time required for maximal activity restoration was about one hour, which was about 500-fold faster than that obtained with C146S. The final specific activity of the mutant mixtures, based on the concentration of R126E, was equivalent to that of the wild-type TS (WT-TS). However, when the activities of E. coli WT-TS and mutant TS mixtures were compared for their extents of renaturation following denaturation as described for Lactobacillus casei TS [Pookanjanatavip, M., Yuthavong, Y., Greene, P. J., & Santi, D. V. (1992) Biochemistry 31, 10303-10309], only about one-half of the activity of WT-TS was restored, implying that the denaturation-renaturation procedure was less efficient than allowing the native TS mutant dimers to exchange subunits. If, as proposed, subunit exchange is responsible for the observed restoration of activity to the E. coli mutant TS mixtures, it would suggest that only one active site cysteine, that provided by R126E in the dimer (R126E)-(C146W), is sufficient to yield the same kcat as WT-TS, which contains one active site cysteine in each subunit. Other mutant dimers that contain both active site cysteines such as (R126E)-(Y94A) and (R126E)-(I264Am) are also fully active, even though one of the subunits is functionally inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…The Cys at amino acid 490 (Fig. 3) is the probable binding site for FdUMP and dUMP (27,37,42,44) and has been proposed as the essential nucleophilic catalyst The alignments used to calculate shared amino acids are shown in Fig. 3.…”

Section: Oligonucleotides a 29-base Oligonucleotide Mixture 5' D[gg(mentioning

confidence: 99%