Differences in natural ligand and fluoropyrimidine binding to human thymidylate synthase identified by transient-state spectroscopic and continuous variation methods

Felder, Takita; Dunlap, R. Bruce; Dix, Daniel B.; Spencer, T. S.

doi:10.1016/s0167-4838(02)00289-3

Cited by 8 publications

(10 citation statements)

References 34 publications

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“…This value is similar to the previously reported values of 1.31 (23). The proposed mechanism of hTS inhibition by PDPA via stabilization of the inactive conformation was expected to lead to non-competitive inhibition since both substrates binding sites in the inactive conformer are affected.…”

Section: Activity Assays and Kinetic Study Of Inhibition By Pdpasupporting

confidence: 91%

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Cooperative Inhibition of Human Thymidylate Synthase by Mixtures of Active Site Binding and Allosteric Inhibitors^,

2007

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Thymidylate synthase (TS) is a target in the chemotherapy of colorectal cancer and some other neoplasms. It catalyses the transfer of a methyl group from methylenetrahydrofolate to dUMP to form dTMP. Based on structural considerations, we have introduced 1,3-propanediphosphonic acid (PDPA) as an allosteric inhibitor of human TS (hTS); it is proposed that PDPA acts by stabilizing an inactive conformer of loop [181][182][183][184][185][186][187][188][189][190][191][192][193][194][195][196][197]. Kinetic studies showed that PDPA is a mixed (noncompetitive) inhibitor vs dUMP. In contrast, vs methylenetrahydrofolate at concentrations lower than 0.25 μM PDPA is an uncompetitive inhibitor, while at PDPA concentrations higher than 1 μM the inhibiton is noncompetive, as expected. At the concentrations corresponding to uncompetitive inhibition, PDPA shows positive cooperativity with an antifolate inhibitor, ZD9331, which binds to the active conformer. PDPA binding leads to the formation of hTS tetramers, but not higher oligomers. These data are consistent with a model in which hTS exists preferably as an asymmetric dimer with one subunit in the active conformation of loop [181][182][183][184][185][186][187][188][189][190][191][192][193][194][195][196][197] and the other in the inactive conformation.Thymidylate synthase (TS) catalyzes the reaction in which the nucleotide deoxyuridylate (dUMP) is reductively methylated by the folate co-substrate 5,10-methylenetetrahydrofolate (CH 2 H 4 folate) to form thymidylate (TMP) and dihydrofolate (1). Substrates are bound in an ordered manner, with dUMP binding at the active site prior to CH 2 H 4 PteGlu. A cysteine residue (Cys195 in hTS) at the active site attacks the 6-position of the pyrimidine base of the nucleotide, resulting in the formation of a covalent bond between TS and the nucleotide and activating the 5-position of the nucleotide for subsequent covalent-bond formation with the C-11 substituent of CH 2 H 4 folate (reviewed in 2-4). The enzyme is the sole source of de novo synthesized thymidylate and its inhibition leads to apoptosis of rapidly dividing cells such as cancer cells, † This work was supported by NIH Grant CA 76560 and the South Carolina Cancer Center. ‡ The PDB file with atomic coordinates of hTS complex with propane-1,3-bisphosphonate have been deposited in the Protein Data Bank as entry xxxx. Data were collected at the Southeast Regional Collaborative Access Team (SER-CAT) 22-BM beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. *To whom correspondence should be addressed at the Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter St., Columbia, South Carolina 29208. Phone (803) 777-2140; fax (803) 777-9521; e-mail lebioda@mail.chem.sc.edu. 1 Abbreviations: TS, thymidylate synthase; ...

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Section: Activity Assays and Kinetic Study Of Inhibition By Pdpasupporting

confidence: 91%

“…Enzyme activity was determined spectrophotometrically as previously described (22,23 2). One unit of enzyme activity is defined as the amount of enzyme required to synthesize 1 μmol of dTMP per minute.…”

Section: Activity Assaysmentioning

confidence: 99%

Cooperative Inhibition of Human Thymidylate Synthase by Mixtures of Active Site Binding and Allosteric Inhibitors^,

2007

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“…Trp fluorescence was used to observe the antifolate binding regarding to the Trp residues at the active site (W80 and W83) (Anderson, O'Neil, DeLano & Stroud, 1999;Felder, Dunlap, Dix & Spencer, 2002;Lovelace, Gibson & Lebioda, 2007;Sharma & Kisliuk, 1975). Samples were prepared by dialysis as described above for ITC.…”

Section: Tryptophan Fluorescencementioning

confidence: 99%

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant

Arvizu‐Flores

Sugich-Miranda

Arreola

et al. 2008

The International Journal of Biochemistry & Cell Biology

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Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH 2 THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen-bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k cat of the K48Q mutant was 430 fold lower than wild-type TS in activity, while the the K m for the (R)-stereoisomer of CH 2 THF was 300 µM, about 30 fold larger than K m from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideaza folate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutamt, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH 2 THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.

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“…The presence of 50 mM peptide reduced the rate of consumption of CH 2 H 4 F Fluorescence studies to probe molecular mechanism of peptide inhibition Earlier structural and fluorescence studies with human TS established that the enzyme undergoes conformational switching between active and inactive states dependent on ligand binding. [26][27][28][29][30][31] The observation that the peptides were able to inhibit T. gondii TS-DHFR in an apo-specific manner, similar to what has been observed for human TS, raised the question of whether T. gondii TS might also utilize an analogous conformational switching to couple active and inactive conformational states. Conformational switching involves a change of position of a TS active site loop (residues 181-197 in human cor-responding to residues 475-491 in T. gondii), which contains the catalytic cysteine, Cys489, that would modulate transitions between active and inactive conformations ( Fig.…”

Section: Steady-state and Pre-steady-state Analysis Of Peptide Inhibimentioning

confidence: 60%

“…Earlier structural and fluorescence studies with human TS established that the enzyme undergoes conformational switching between active and inactive states dependent on ligand binding . The observation that the peptides were able to inhibit T. gondii TS–DHFR in an apo‐specific manner, similar to what has been observed for human TS, raised the question of whether T. gondii TS might also utilize an analogous conformational switching to couple active and inactive conformational states.…”

Section: Resultsmentioning

confidence: 99%

Selective peptide inhibitors of bifunctional thymidylate synthase‐dihydrofolate reductase from Toxoplasma gondii provide insights into domain–domain communication and allosteric regulation

2013

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The bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) plays an essential role in DNA synthesis and is unique to several species of pathogenic protozoans, including the parasite Toxoplasma gondii. Infection by T. gondii causes the prevalent disease toxoplasmosis, for which TS-DHFR is a major therapeutic target. Here, we design peptides that target the dimer interface between the TS domains of bifunctional T. gondii TS-DHFR by mimicking bstrands at the interface, revealing a previously unknown allosteric target. The current study shows that these b-strand mimetic peptides bind to the apo-enzyme in a species-selective manner to inhibit both the TS and distal DHFR. Fluorescence spectroscopy was used to monitor conformational switching of the TS domain and demonstrate that these peptides induce a conformational change in the enzyme. Using structure-guided mutagenesis, nonconserved residues in the linker between TS and DHFR were identified that play a key role in domain-domain communication and in peptide inhibition of the DHFR domain. These studies validate allosteric inhibition of apo-TS, specifically at the TS-TS interface, as a potential target for novel, species-specific therapeutics for treating T. gondii parasitic infections and overcoming drug resistance.

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Differences in natural ligand and fluoropyrimidine binding to human thymidylate synthase identified by transient-state spectroscopic and continuous variation methods

Cited by 8 publications

References 34 publications

Cooperative Inhibition of Human Thymidylate Synthase by Mixtures of Active Site Binding and Allosteric Inhibitors^,

Cooperative Inhibition of Human Thymidylate Synthase by Mixtures of Active Site Binding and Allosteric Inhibitors^,

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant

Selective peptide inhibitors of bifunctional thymidylate synthase‐dihydrofolate reductase from Toxoplasma gondii provide insights into domain–domain communication and allosteric regulation

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Differences in natural ligand and fluoropyrimidine binding to human thymidylate synthase identified by transient-state spectroscopic and continuous variation methods

Cited by 8 publications

References 34 publications

Cooperative Inhibition of Human Thymidylate Synthase by Mixtures of Active Site Binding and Allosteric Inhibitors,

Cooperative Inhibition of Human Thymidylate Synthase by Mixtures of Active Site Binding and Allosteric Inhibitors,

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant

Selective peptide inhibitors of bifunctional thymidylate synthase‐dihydrofolate reductase from Toxoplasma gondii provide insights into domain–domain communication and allosteric regulation

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Resources

About

Cooperative Inhibition of Human Thymidylate Synthase by Mixtures of Active Site Binding and Allosteric Inhibitors^,

Cooperative Inhibition of Human Thymidylate Synthase by Mixtures of Active Site Binding and Allosteric Inhibitors^,