Direct Spectrophotometric Evidence for the Oxidation of Tetrahydrofolate during the Enzymatic Synthesis of Thymidylate

Wahba, Amy; Friedkin, Morris

doi:10.1016/s0021-9258(18)64341-8

Cited by 219 publications

(45 citation statements)

References 10 publications

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“…PcTS was expressed and puri¢ed as described previously [33]. Enzyme activity was assayed spectrophotometrically according to the methods of Wahba and Friedkin [34]. Inhibition assays measured the initial velocities of reactions with 1.9 Wg enzyme, 80 WM dUMP, 100 WM mTHF and varying concentrations of inhibitor.…”

Section: Expression and Assaysmentioning

confidence: 99%

Approaches to solving the rigid receptor problem by identifying a minimal set of flexible residues during ligand docking11PDB coordinates have been deposited with the RSCB with accession ID: 1F28.

Anderson

O'Neil

Surti

et al. 2001

Chemistry & Biology

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Based on the examination of many protein/ligand complexes, we develop an algorithm (SOFTSPOTS) for identifying regions of a protein target that are more likely to accommodate plastically to regions of a drug molecule. Using these indicators we develop a second algorithm (PLASTIC) that provides a minimal manifold of possible conformations of a protein target for drug design, reducing the bias in structure-based drug design imparted by structures of enzymes co-crystallized with inhibitors.

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Section: Expression and Assaysmentioning

confidence: 99%

Approaches to solving the rigid receptor problem by identifying a minimal set of flexible residues during ligand docking11PDB coordinates have been deposited with the RSCB with accession ID: 1F28.

Anderson

O'Neil

Surti

et al. 2001

Chemistry & Biology

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“…TS activity was monitored spectrophotometrically by the increase in absorbance at 340 nm that occurs concomitant with the production of H 2 -folate (∆ε = 6400 M -1 cm -1 ) (16,17). To the standard reaction buffer was added 300 µM of the racemic mixture (150 µM of the active isomer) of CH 2 H 4 -folate and 200 µM dUMP.…”

Section: Ts Enzyme Assaysmentioning

confidence: 99%

Tolerance of 5-fluorodeoxyuridine resistant human thymidylate synthases to alterations in active site residues

Landis

Gerlach

Adman

et al. 1999

Nucleic Acids Research

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Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. In the cell, 5-FU is metabolized to 5-fluorodeoxyuridylate (5-FdUMP), a tight binding covalent inhibitor of thymidylate synthase (TS). In order to create 5-FdUMP resistant enzymes to protect chemosensitive normal cells and further understand mechanisms of 5-FdUMP resistance, we have randomized four residues within the active site of TS. Our previous studies identified alterations in residues which produce active TS with enhanced resistance to 5-fluorouridine (5-FdUR). By remutagenizing a subset of the 13 previously targeted residues (A197, L198, C199 and V204), an unbiased random library can be created allowing for extensive testing of all possible amino acid substitutions at each of the sites. Using genetic complementation and selection in Escherichia coli, we identified the spectrum of substitutions that yield active TS as well as those that resulted in 5-FdUR resistant mutants of TS. The 5-FdUR resistant TS were found to share several structural features including hydrophobic substitutions at residue 197, retention of the wild-type leucine 198, the alteration C199L (present in 64% of the drug-resistant library), and polar alterations of valine 204. The catalytic activity of mutants with these features was approximately equal to that of the wild-type TS.

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“…Further, using limiting 3-MedUMP as substrate, there was an increase in absorbance at 340 nm which corresponded to ∆ max (340 nm) of 6590 M -1 cm -1 . This differential extinction coefficient is characteristic of the conversion of CH 2 H 4 folate to H 2 folate [∆ max (340 nm) of 6400 M -1 cm -1 ] (Wahba, 1961). Together, these data provide convincing evidence that TS N229C catalyzes the reaction of 3-Me-dUMP with CH 2 H 4 folate to produce 3-MedTMP and H 2folate.…”

Section: Resultsmentioning

confidence: 68%

Asparagine 229 Mutants of Thymidylate Synthase Catalyze the Methylation of 3-Methyl-2‘-deoxyuridine 5‘-Monophosphate

Costi¹,

Liu²,

Finer-Moore³

et al. 1996

Biochemistry

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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 2'-deoxyuridine 5'-monophosphate (dUMP). Asn 229 is not essential for substrate binding or catalysis [Liu, l., & Santi, D. B. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8604-8608] but is a major determinant in substrate specificity [Liu, l., & Santi, D. V. (1993) Biochemistry 32, 9263-9267]. 3-Methyl-dUMP (3-MedUMP) is neither a substrate nor an inhibitor of wild type TS but is converted to 3-methyl 2'-deoxythymidine 5'-monophosphate by many TS Asn 229 mutants. Some of the Asn 229 mutants (N229C, -I, -M, -A, and -V) have kcat values for 3-MedUMP methylation which are up to about 20% of that for wild type TS-catalyzed methylation of dUMP, and some mutants (N229C and -A) catalyze methylation of 3-MedUMP more efficiently than that of dUMP. Mutants with hydrophobic side chains tended to be more active in catalysis of methylation of 3-MedUMP than those with hydrophilic side chains. The ability of 3-MedUMP to serve as a substrate for Asn 229 mutants shows that the active form of dUMP involves the neutral pyrimidine base and that ionization of the 3-NH group does not occur in the course of catalysis. In contrast to the negligible binding of 3-MedUMP to wild type TS, both 3-MedUMP and dUMP showed similar Km values with the Asn 229 mutants, suggesting similar binding affinities to the mutants. The X-ray crystal structure of the TS N229C--3-MedUMP complex showed that the side chain of Cys 229 was rotated away from the pyrimidine ring to allow placement of a water molecule and the 3-methyl group of 3-MedUMP in the active site. Our results suggest that the inability of 3-MedUMP to undergo methylation by wild type TS is due to its inability to bind to the enzyme, which in turn is simply a result of steric interference of the 3-methyl group with the side chain of Asn 229.

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Direct Spectrophotometric Evidence for the Oxidation of Tetrahydrofolate during the Enzymatic Synthesis of Thymidylate

Cited by 219 publications

References 10 publications

Approaches to solving the rigid receptor problem by identifying a minimal set of flexible residues during ligand docking11PDB coordinates have been deposited with the RSCB with accession ID: 1F28.

Approaches to solving the rigid receptor problem by identifying a minimal set of flexible residues during ligand docking11PDB coordinates have been deposited with the RSCB with accession ID: 1F28.

Tolerance of 5-fluorodeoxyuridine resistant human thymidylate synthases to alterations in active site residues

Asparagine 229 Mutants of Thymidylate Synthase Catalyze the Methylation of 3-Methyl-2‘-deoxyuridine 5‘-Monophosphate

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