Thymidylate synthetase catalyzed exchange of tritium from [5-3H]-2'-deoxyuridylate for protons of water

Pogolotti, Alfonso L.; Weill, C. Edwin; Santi, Daniel V.

doi:10.1021/bi00580a016

Cited by 46 publications

(37 citation statements)

References 16 publications

(21 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Given the evidence that TSase can form a covalent bond between the sulfur atom of C146 and C6 of dUMP even in the absence of cofactor (41), and the bond cannot form in the C146S mutant, an important question is whether this defect is responsible for the apparent weaker binding affinity. To consider this, we turned to 1 H- 13 C ILV methyl HSQC spectra of the wild - type dUMP-bound complex, in which we detect a minor state that is likely the covalent complex based on the following: 1) The minor resonances are not present in C146S-dUMP spectra and the single set of resonances in the mutant spectrum overlap with the major state in the wild-type spectrum (Figure 2A).…”

Section: Resultsmentioning

confidence: 99%

Widespread Perturbation of Function, Structure, and Dynamics by a Conservative Single-Atom Substitution in Thymidylate Synthase

Sapienza

Lee

2016

Biochemistry

View full text Add to dashboard Cite

Thymidylate synthase (TSase) is responsible for synthesizing the sole de novo source of dTMP in all organisms. TSase is a drug target and as such it is well studied both in terms of structure and reaction mechanism. Cysteine 146 in E. coli TSase is universally conserved because it serves as the nucleophile in the enzyme mechanism. Here we use the C146S mutation to probe the role of the sulfur atom in early events in the catalytic cycle beyond serving as the nucleophile. Surprisingly, the single atom substitution severely decreases substrate binding affinity, and the unfavorable ΔΔGbind° is comprised of roughly equal enthalpic and entropic components at 25 °C. Chemical shifts in the free and dUMP-bound states show the mutation causes perturbations throughout TSase, including regions important for complex stability, in agreement with a less favorable enthalpy change. We measured the NMR methyl symmetry axis order parameter (S2axis), a proxy for conformational entropy, for TSase at all vertices of the dUMP binding/C146S mutation thermodynamic cycle and found that the calculated TΔΔS°conf is of similar sign and magnitude as the calorimetric TΔΔS°. Further, we ascribed minor resonances in wild-type-dUMP spectra to a state with a covalent bond between the Sγ of C146 and C6 of dUMP, and find S2axis values are unaffected by covalent bond formation, indicating this reaction step is neutral with respect to ΔS°conf. Lastly, the C146S mutation enabled us to measure cofactor analog binding by ITC without the confounding heat signature of covalent bond formation. Raltitrexed binds free and singly bound TSase with similar affinities, yet the two binding events have different enthalpy changes, providing further evidence of communication between the two active sites.

show abstract

Section: Resultsmentioning

confidence: 99%

Widespread Perturbation of Function, Structure, and Dynamics by a Conservative Single-Atom Substitution in Thymidylate Synthase

Sapienza

Lee

2016

Biochemistry

View full text Add to dashboard Cite

show abstract

“…(9), except that the buffer used was 50 mM Tes, pH 7.4/5 mM dithiothreitol/1 mM EDTA/25 mM MgCl2. DHFR catalytic activity was measured spectrophotometrically at 25°C (10) in 1.0 ml of a mixture containing 0.1 mM H2 folate, 0.1 mM NADPH, 50 mM Tes (pH 7.4), 75 mM 2-mercaptoethanol, and 1 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica.

Coderre¹,

Beverley²,

Schimke³

et al. 1983

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

192

View full text Add to dashboard Cite

Leishmania tropica promastigotes that are highly resistant to methotrexate, a dihydrofolate reductase inhibitor, have been developed. Organisms resistant to 1 mM methotrexate have a 40-fold increase in dihydrofolate reductase which is associated with thymidylate synthetase, and they contain amplified regions of DNA that may be directly visualized on stained gels of restriction digests. The amplified DNA in these organisms is about 56 Idlobases in length, has a copy number about 80-fold higher than that of wild-type organisms, and constitutes about 10% of the nuclear DNA. When the methotrexate-resistant L. tropica are propagated in drug-free medium, the dihydrofolate reductase-thymidylate synthetase protein and the amplified DNA decrease in a parallel fashion until they are indistinguishable from the levels in wild-type organisms. However, when these apparent revertants are again challenged with 1 mM methotrexate, enzyme overproduction and DNA amplification occur rapidly. As in mammalian cells, it appears that drug resistance in parasitic protozoa may be mediated by gene amplification.Drug resistance in protozoan parasites was first described early in this century by Paul Ehrlich in his studies on the chemotherapy of trypanosomiasis (1). Since then, numerous instances of drug-resistant Trypanosoma (2, 3), Leishmania (3), and Plasmodium (4) have been documented. There are relatively few drugs that are curative agents for the diseases caused by pathogenic protozoa, and the emergence of drug-resistant organisms is a particularly serious problem. Furthermore, because knowledge of the mechanisms of drug resistance in protozoan parasites is limited, no rational approaches exist for circumventing or avoiding this problem.We have embarked upon investigations to define the molecular bases of drug resistance in pathogenic protozoa. In this report, we describe the development and properties of methotrexate (MTX)-resistant strains of Leishmania tropica promastigotes. We show that resistant organisms overproduce the drug target-a bifunctional dihydrofolate reductase (DHFR)-thymidylate synthetase (TS) enzyme-and amplify certain regions of DNA. These properties are unstable when selective pressure of MTX is removed but rapidly reemerge when organisms are reexposed to normally lethal concentrations of the drug. Last, we show that only a 10-fold increase in gene copy number in these protozoa may be detected by direct visualization of ethidium bromide-stained gels of DNA restriction digests.MATERIALS EC50 values refer to the concentration of MTX that inhibited the growth rate by 50%; unless otherwise specified, one passage refers to five generations of growth, or a 32-fold expansion in cell number, followed by reseeding in fresh medium. Cells were counted by using a Coulter Counter ZBI.Selection of MTX-Resistant Cel Lines. MTX-resistant strains of L. tropica promastigotes were obtained by using a stepwise selection process. Cells were seeded at approximately 106/ml in medium containing the specified amount of MTX. When th...

show abstract

“…The purified enzymes were concentrated and desalted using Amicon Centriprep-30 concentrators and stored at -80°C. monitored by the increase in absorbance at 340 nm associated with the formation of dihydrofolate (∆ ) 6400 M -1 cm -1 ) (18) (19). The reaction mixture contained 600 µM 6(R)-CH 2 H 4 folate and 0.1-3.0 mM [2-14 C, 5-3 H] dUMP along with 5-13 µM enzyme in the standard TES assay buffer.…”

Section: Methodsmentioning

confidence: 99%

A Novel dCMP Methylase by Engineering Thymidylate Synthase

et al. 1997

Self Cite

View full text Add to dashboard Cite

X-ray crystal structures of binary complexes of dUMP or dCMP with the Lactobacillus casei TS mutant N229D, a dCMP methylase, revealed that there is a steric clash between the 4-NH 2 of dCMP and His 199, a residue which normally H-bonds to the 4-O of dUMP but is not essential for activity. As a result, the cytosine moiety of dCMP is displaced from the active site and the catalytic thiol is moved from the C6 of the substrate about 0.5 Å further than in the wild-type TS-dUMP complex. We reasoned that combining the N229D mutation with mutations at residue 199 which did not impinge on the 4-NH 2 of dCMP should correct the displacements and further favor methylation of dCMP. We therefore prepared several TS N229D mutants and characterized their steady state kinetic parameters. TS H199A/N229D showed a 10 11 change in specificity for methylation of dCMP Versus dUMP. The structures of TS H199A/ N229D in complex with dCMP and dUMP confirmed that the position and orientation of bound dCMP closely approaches that of dUMP in wild-type TS, whereas dUMP was displaced from the optimal catalytic binding site.Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of 2′-deoxyuridine-5′-monophosphate (dUMP) by 5,10-methylene-5,6,7,8-tetrahydrofolate (CH 2 H 4 -folate) to form deoxythymidine-5′-monophosphate (dTMP) and 7,8-dihydrofolate (H 2 folate). Comparison of sequences of TS from ∼30 different organisms reveals that it is one of the most highly conserved enzymes (1, 2). A large number of three-dimensional structures of free and ligand-bound TSs and TS mutants have been determined and provide a structural understanding of substrate recognition, stereochemical aspects of the reaction pathway (3), and roles of specific residues in the reaction chemistry (4).Asn 229 is invariant in all TS's sequenced to date, and forms a cyclic hydrogen bond network with the 4-O and 3-NH of the substrate dUMP (Figure 2a) (5-7). Nevertheless, it can be replaced by numerous other residues, and the resultant mutants retain TS activity and also gain the capability to bind 2′-deoxycytidine 5′-monophosphate (dCMP) tightly (8,9). Moreover, alteration of Asn 229 to aspartate provides an enzyme which methylates dCMP more efficiently than dUMP, although the catalytic efficiency is still significantly lower than is the wild-type TS for its natural substrate dUMP (10, 11).

show abstract

Thymidylate synthetase catalyzed exchange of tritium from [5-3H]-2'-deoxyuridylate for protons of water

Cited by 46 publications

References 16 publications

Widespread Perturbation of Function, Structure, and Dynamics by a Conservative Single-Atom Substitution in Thymidylate Synthase

Widespread Perturbation of Function, Structure, and Dynamics by a Conservative Single-Atom Substitution in Thymidylate Synthase

Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica.

A Novel dCMP Methylase by Engineering Thymidylate Synthase

Contact Info

Product

Resources

About