Kinetics and mechanism of interaction of 10-propargyl-5,8-dideazafolate with thymidylate synthase

Pogolotti, Alfonso L.; Danenberg, Peter V.; Santi, Daniel V.

doi:10.1021/jm00154a009

Cited by 94 publications

(62 citation statements)

References 5 publications

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“…The purification procedures of wild-type and mutant TSs were carried out as described previously (30,32). TS activity was monitored by a spectrophotometric assay as described previously (33,34). Methods for measurement of k cat values, the Michaelis constants (K m values) for CH 2 H 4 folate and dUMP and inhibition constants (K i values) for raltitrexed, Thymitaq, BW1843U89, and FdUMP were as described previously (28).…”

Section: Methodsmentioning

confidence: 99%

Probing the Folate-binding Site of Human Thymidylate Synthase by Site-directed Mutagenesis

Tong¹,

Liu-Chen²,

Ercikan-Abali³

et al. 1998

Journal of Biological Chemistry

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Human thymidylate synthase (TS) contains three highly conserved residues Ile-108, Leu-221, and Phe-225 that have been suggested to be important for cofactor and antifolate binding. To elucidate the role of these residues and generate drug-resistant human TS mutants, 14 variants with multiple substitutions of these three hydrophobic residues were created by site-directed mutagenesis and transfected into mouse TS-negative cells for complementation assays and cytotoxicity studies, and the mutant proteins expressed and characterized. The I108A mutant confers resistance to raltitrexed and Thymitaq with respective IC 50 values 54-and 80-fold greater than wild-type but less resistance to BW1843U89 (6-fold). The F225W mutant displays resistance to BW1843U89 (17-fold increase in IC 50 values), but no resistance to raltitrexed and Thymitaq. It also confers 8-fold resistance to fluorodeoxyuridine. Both the kinetic characterization of the altered enzymes and formation of antifolate-resistant colonies in mouse bone marrow cells that express mutant TS are in accord with the IC 50 values for cytotoxicity noted above. The human TS mutants (I108A and F225W), by virtue of their desirable properties, including good catalytic function and resistance to antifolate TS inhibitors, confirm the importance of amino acid residues Ile-108 and Phe-225 in the binding of folate and its analogues. These novel mutants may be useful for gene transfer experiments to protect hematopoietic progenitor cells from the toxic effects of these drugs.Thymidylate synthase (TS), 1 which catalyzes the conversion of dUMP to dTMP, is an attractive target for drug design (1-5). TS inhibitors, which occupy either the substrate or cofactorbinding site, have been designed based on the structure and properties of the enzyme. Fluoropyrimidines, such as 5-fluorouracil (5-FU) and fluorodeoxyuridine (FdUrd), are metabolized to 5-fluoro-2-deoxyuridine monophosphate (FdUMP) and compete subsequently with the substrate, dUMP, for its binding site and have been used in the clinic for over 40 years to treat breast and gastrointestinal cancers. However, fluoropyrimidines, due to their incorporation into DNA and RNA, are not pure TS inhibitors. Also, they are susceptible to metabolic degradation in vivo. In contrast, the cofactor CH 2 H 4 folate is a relatively large molecule and has a variety of binding sites that may be altered in drug design. In recent years folate analogues have been designed as highly specific and stable TS inhibitors (6). The inhibitor CB3717 was the first folate analogue inhibitor of TS tested in the clinic and although anti-tumor activity was demonstrated, its further development was abandoned due to renal and hepatic toxicity (7,8). The information provided by the crystal structures of TS from bacterial and mammalian sources (9 -18) has led to the design and synthesis of novel analogues of CH 2 H 4 folate, e.g. raltitrexed (Tomudex ® , ZD1694), BW1843U89, Thymitaq (AG337), and AG331 (19 -27). These new and promising agents have entered clinical trials...

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Section: Methodsmentioning

confidence: 99%

Probing the Folate-binding Site of Human Thymidylate Synthase by Site-directed Mutagenesis

Tong¹,

Liu-Chen²,

Ercikan-Abali³

et al. 1998

Journal of Biological Chemistry

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“…Enzymatic activity was monitored by spectrophotometric assay of the conversion of CH2H4folate to H2folate under previously described conditions (Pogolotti et al, 1986). Assay solutions contained 40 pM dUMP, 100 pM CH2H4folate, and between 0.1 and 0.5 pM enzyme.…”

Section: Methodsmentioning

confidence: 99%

Reversible dissociation and unfolding of the dimeric protein thymidylate synthase

et al. 1992

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Conditions for in vitro unfolding and refolding of dimeric thymidylate synthase from Lactobacillus casei were found. Ultraviolet difference and circular dichroism spectra showed that the enzyme was completely unfolded at concentrations of urea over 5.5 M. As measured by restoration of enzyme activity, refolding was accomplished when 0.5 M potassium chloride was included in the refolding mixture. Recombination of subunits from catalytically inactive mutant homodimers to form an active hybrid dimer was achieved under these unfolding-refolding conditions, demonstrating a monomer to dimer association step.Keywords: dimerization; folding; oligomerization; thymidylate synthase Thymidylate synthase (TS) is a dimer of two identical, 35-kDa subunits (Kinemage 1) that catalyzes the conversion of deoxyuridine monophosphate (dUMP) and 5,lO-methylenetetrahydrofolate (CH2H4folate) to dTMP and 7,8-dihydrofolate (H2folate). TS provides the only de novo pathway to deoxythymidine monophosphate (dTMP) production and has been studied extensively from the standpoints of structure, function, and inhibition (Santi & Danenberg, 1984). The crystal structure, which has been determined to 2.3 A resolution for Lactobacillus casei TS (Hardy et al., 1987;Perry et al., 1990; Finer-Moore, unpubl.), shows that the monomers form dimer contacts primarily between two, five-stranded beta sheets that maintain a unique +28" dihedral angle between strand directions (Kinemage 4). Three of these strands form abeta kink that creates part of the active site pocket of the second monomer. Further, two active site arginine residues (178' and 179') are donated from the opposing monomer and coordinate with the phosphate moiety of the substrate (Kinemage 3). Thus, the dimeric structure of TS is essential for the function of the enzyme.In order to study interactions at the dimer interface of TS, we sought conditions that would allow the separation, unfolding, and reassembly of subunits. To date, there is no evidence that the dimer can be dissociated and reformed. In the present work, we show that TS can be denatured to unfolded monomers in urea and subsequently refolded to its catalytically active native state. Results and discussionThe concentration of urea required to unfold TS was determined using UV and CD spectroscopies. The difference in environment of tryptophan residues between native and unfolded protein was monitored by the UV absorbance change at 294 nm (Fig. 1). Three zones were apparent along the equilibrium unfolding transition: (1) a range below 3.5 M urea where spectral changes were not observed, (2) a sharp transition between 3.5 M and 5.5 M denaturant, and (3) a range above 5.5 M urea where no further change in absorbance was detected. The AGHZ0 calculated for this equilibrium unfolding transition at 0 M urea was 19.1 kcal mol" (see Materials and methods). To ascertain whether secondary structure was lost at high urea concentration, the CD spectrum of TS equilibrated in 8.0 M urea was compared with that of native protein (Fig. 2). With...

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“…That these interactions are sufficiently strong to overcome repulsion between Glu 126' and the dUMP phosphate, as well as to induce the closed protein conformation, points to the usefulness of quinazolinebased TS inhibitors. Indeed, CB3717 has a nanomolar inhibitory constant and an extremely long association half-life (Pogolotti et al, 1986). However, the loss of covalent bond formation in one active site suggests the mutant protein is less able to achieve covalent intermediate I11 (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…We have recently suggested that the poor density observed for the covalent bond between Cys 146 and nucleotide in this and other complexes may reflect a weak or polarized bond during catalysis that is required to help the enzyme I rotate out of the active site to relieve electrostatic repulsion with the dUMP phosphate, yet the R126E mutant was -2000-fold less active than wild-type TS. Two crystal structures of R126E were obtained: the first from crystals grown in the presence of dUMP, and the second in the presence of dUMP and CB3717, an analog of CH2THF that displays a nanomolar inhibition constant (Jones et al, 1981;Pogolotti et al, 1986). In both proteins, Glu 126' occupied the same position as Arg 126' in the wild-type protein, resulting in not only electrostatic, but also steric interference with the dUMP phosphate.…”

Section: I1mentioning

confidence: 99%

Crystal structures of a marginally active thymidylate synthase mutant, Arg 126 → Glu

Strop¹,

Changchien²,

Maley³

et al. 1997

Protein Science

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Thymidylate synthase (TS) is a long-standing target for anticancer drugs and is of interest for its rich mechanistic features. The enzyme catalyzes the conversion of dUMP to dTMP using the co-enzyme methylenetetrahydrofolate, and is perhaps the best studied of enzymes that catalyze carbon-carbon bond formation. Arg 126 is found in all TSs but forms only 1 of 13 hydrogen bonds to dUMP during catalysis, and just one of seven to the phosphate group alone. Despite this, when Arg 126 of TS from Escherichia coli was changed to glutamate (R126E), the resulting protein had kc,, reduced 2000-fold and K , reduced 600-fold. The crystal structure of R126E was determined under two conditions-in the absence of bound ligand (2.4 P\ resolution), and with dUMP and the antifolate CB3717 (2.2 8, resolution). The first crystals, which did not contain dUMP despite its presence in the crystallization drop, displayed Glu 126 in a position to sterically and electrostatically interfere with binding of the dUMP phosphate. The second crystals contained both dUMP and CB3717 in the active site, but Glu 126 formed three hydrogen bonds to nearby residues (two through water) and was in a position that partially overlapped with the normal phosphate binding site, resulting in a -1 8, shift in the phosphate group. Interestingly, the protein displayed the typical ligand-induced conformational change, and the covalent bond to Cys 146 was present in one of the protein's two active sites.

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Kinetics and mechanism of interaction of 10-propargyl-5,8-dideazafolate with thymidylate synthase

Cited by 94 publications

References 5 publications

Probing the Folate-binding Site of Human Thymidylate Synthase by Site-directed Mutagenesis

Probing the Folate-binding Site of Human Thymidylate Synthase by Site-directed Mutagenesis

Reversible dissociation and unfolding of the dimeric protein thymidylate synthase

Crystal structures of a marginally active thymidylate synthase mutant, Arg 126 → Glu

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