Mechanism of interaction of thymidylate synthetase with 5-fluorodeoxyuridylate (original) (raw)
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Thymidylate synthase-catalyzed, tetrahydrofolate-dependent self-inactivation by 5-FdUMP
Archives of Biochemistry and Biophysics, 2019
In view of previous crystallographic studies, N 4-hydroxy-dCMP, a slow-binding thymidylate synthase inhibitor apparently caused "uncoupling" of the two thymidylate synthase-catalyzed reactions, including the N 5,10-methylenetetrahydrofolate one-carbon group transfer and reduction, suggesting the enzyme's capacity to use tetrahydrofolate as a cofactor reducing the pyrimidine ring C(5) in the absence of the 5-methylene group. Testing the latter interpretation, a possibility was examined of a TS-catalyzed covalent self-modification/self-inactivation with certain pyrimidine deoxynucleotides, including 5-fluoro-dUMP and N 4-hydroxy-dCMP, that would be promoted by tetrahydrofolate and accompanied with its parallel oxidation to dihydrofolate. Electrophoretic analysis showed mouse recombinant TS protein to form, in the presence of tetrahydrofolate, a covalently bound, electrophoretically separable 5-fluoro-dUMP-thymidylate synthase complex, similar to that produced in the presence of N 5,10-methylenetetrahydrofolate. Further studies of the mouse enzyme binding with 5-fluoro-dUMP/ N 4-hydroxy-dCMP by TCA precipitation of the complex on filter paper showed it to be tetrahydrofolate-promoted, as well as to depend on both time in the range of minutes and the enzyme molecular activity, indicating thymidylate synthase-catalyzed reaction to be responsible for it. Furthermore, the tetrahydrofolate-and timedependent, covalent binding by thymidylate synthase of each 5-fluoro-dUMP and N 4-hydroxy-dCMP was shown to be accompanied by the enzyme inactivation, as well as spectrophotometrically confirmed dihydrofolate production, the latter demonstrated to depend on the reaction time, thymidylate synthase activity and temperature of the incubation mixture, further documenting its catalytic character.
Thymidylate synthase: Structure, inhibition, and strained conformations during catalysis
Pharmacology & Therapeutics, 1997
Thymidylate synthase (TS) is a long-standing target for chemotherapeutic agents because of its central role in DNA synthesis, and it is also of interest because of its rich mechanistic features. The reaction catalyzed by TS is the methylation of dUMP, with the transferred methyl group provided by the cofactor methylenetetrahydrofolate (CH,THF). R ecently, several crystal structure determinations and mechanistic studies have led to a deeper understanding of the TS reaction mechanism, and address the role of conformational change in TS catalysis and inhibition. Included among these structures are complexes of TS bound to substrate dUMP; cofactor CH,THF; the nucleotide analogs ii-fluoro-dUMP, 5-nitro-dUMP and dGMP; and the promising antifolates BW1843, ZD1694, and AG337. From these studies, a picture of TS emerges where ligand-induced conformational changes play key roles in catalysis by straining the thiol adduct that occurs during the reaction; by protecting the highly reactive reaction intermediates; and by providing a means to stabilize a high-energy conformer of the cofactor after initial binding of a low-energy conformer.
Bioorganic & Medicinal Chemistry Letters, 2000
AbstractÐ5-Fluoropropynyl-2 H -deoxyuridine 5 H -phosphate (3) was designed as a mechanism-based inactivator of thymidylate synthase (TS). The inhibitor was synthesized from 5-iodo-2 H -deoxyuridine and propargyl alcohol by palladium-catalyzed coupling, followed by¯uorination and selective phosphorylation. Incubation of TS with 3, in the presence or absence of the CH 2 H 4 folate cofactor, caused rapid, irreversible inactivation of the enzyme. #
Biochemistry, 1997
We have determined kinetic and thermodynamic constants governing binding of substrates and products to thymidylate synthase from Escherichia coli (TS) sufficient to describe the kinetic scheme for this enzyme. (1) The catalytic mechanism is ordered in the following manner, TS + dUMP f TS‚dUMP + (6R)-5,10-CH 2-H 4 folate f TS‚dUMP‚(6R)-5,10-CH 2 H 4 folate f TS‚dTMP‚H 2 folate f TS‚dTMP f TS as predicted previously by others from steady-state measurements. (2)When substrates are saturating, the overall reaction rate is governed by the slow conversion of enzyme-bound substrates to bound products as demonstrated by (i) large primary and secondary isotope effects on k cat and (ii) high rates of product dissociation compared to k cat. (3) Stopped-flow studies measuring the binding of 10propargyl-5,8-dideazafolate, an analog of (6R)-5,10-CH 2 H 4 folate, with the active site mutant C146A or the C-terminus-truncated mutant P261Am enabled us to identify physical events corresponding to spectral changes which are observed with the wild-type enzyme during initiation of catalysis. A kinetically identifiable reaction step, TS‚dUMP‚(6R)-5,10-CH 2 H 4 folate f (TS‚dUMP‚(6R)-5,10-CH 2 H 4 folate)*, likely represents reorientation of the C-terminus of the enzyme over the catalytic site. This seals the substrates into a relatively nonaqueous environment in which catalysis can occur. (4) Although TS is a dimer of identical subunits, catalysis is probably confined to only one subunit at a time. (5) The "high-resolution" kinetic scheme described herein provides a framework for the interpretation of the kinetics of catalysis by mutant ecTS chosen to provide insights into the relationship between structure and function.
Biochemistry, 1990
Amino acid replacements have been introduced in specific sites of bacteriophage T4 thymidylate synthase (TCTS) to assess the role that these changes have on enzyme activity. Each of the conserved amino acids in the active-site region of TCTS was modified, and the effects that these changes had on the kinetic and physical properties of this enzyme were measured. The mutations introduced were Pro-155-Ala (P155A), Cys-156-Ser (C156S), and His-157-Val (H157V) with the resulting synthases possessing kat's of 10.3,0.008, and 2.70 s-I, respectively, relative to that of the wild-type enzyme of 11.8 s-'. Equilibrium dialysis was performed on the wild-type and mutant enzymes to determine the binding constants for 2'-deoxyuridylate and 5-fluoro-2'-deoxyuridylate, and while in most cases the extent of binding of these nucleotides to the mutant proteins was reduced when compared with wild-type TS, the number of binding sites involved remained about 1 or less for the binary complex and almost 2 for the ternary complex. Heat and urea stability studies revealed that the mutant with the highest enzyme activity, P155A, was the most unstable, while spectrofluorometric analyses revealed that the structures of P155A and H157V were perturbed relative to the C156S and wild-type TSs. These studies are in agreement with others implicating the phylogenetically conserved active-site cysteine as playing an essential mechanistic role in the catalytic process promoted by TS. The proximal amino acids on either side of this cysteine, although also highly conserved, do not appear to affect the catalytic mechanism directly, but may do so indirectly through their influence on the conformation at the active site as well as other regions of the enzyme. Amino acids replacements were introduced also into the folate and deoxynucleotide 5'-phosphate binding sites of the T4-phage TS to ascertain the potential role that these amino acids play in the catalytic process. These positions were selected on the basis of previous chemical modification and X-ray crystallographic studies on Lactobacillus casei TS. Amino acid residues 48 and 49, which are in the putative folate binding site, were converted from lysines to arginines; in the former case, the mutated enzyme had less than 7% of the wild-type activity while in the latter, the mutated enzyme still retained about 60% of its activity. Spectrofluorometric studies revealed the K49R T4-TS mutation to affect a conformational change in the enzyme's structure, but little or no change was observed in the spectra of the T4-TS from K48R. The latter enzyme was impaired in its interaction with CH2H4PteGlu, as evidenced by a greater than 3-fold increase in its K,. On the basis of these and our previous folylpolyglutamate fixation studies (Maley et al., 1982), it would appear that Lys-48 of T4-TS (Lys-50 in L. casei TS) contributes to the binding of folate substrates and their analogues to a greater degree than Lys-49 of T4-TS (Lys-51 in L. casei TS). Replacement of Arg-137 and Arg-176 in the phosphate binding sites of TCTS with lysine residues diminishes enzyme activity by 70% in the former case, and almost completely in the latter. The TS from R137K does not show a spectrofluorometric shift, while the synthase from R176K does. However, the mutant enzyme from R137G shows a blue shift in its fluorescence spectrum, which is associated with a complete loss in activity. From these studies, it would appear that while both Arg-137 and Arg-176 promote nucleotide binding, the latter contributes more to this phenomenon than the former. ymid ylate synthase (TS)' (EC 2.1.1.45) catalyzes one of the most unusual reactions in nature wherein CH,H,PteGlu serves as both a one-carbon donor and a reductant in the conversion of dUMP to dTMP (Friedkin et al., 1957). The most plausible mechanism for this reaction involves an initial attack of a nucleophile from the enzyme on the 6-position of dUMP, followed by the directed addition of the methylene group from CH,H,PteGlu to the 5-position of dUMP (Pogolloti & Santi, 1977). Kinetic studies with TSs from both eukaryotic (Lorenson et al., 1967) and prokaryotic (Daron & Aull, 1978) sources, as well as substrate binding studies (Galivan et al., 1976a,b), are in agreement with this sequential
Human thymidylate synthetase—III
Biochemical Pharmacology, 1979
The structure-activity relationship of human thymidylate synthetase (EC 2.1.1.45) was studied with two groups of folate analogs: (I) methotrexate (MTX) analogs modified at the glutamate residue and N": and (2) tetrahydrofolate (H,PteGlu) analogs modified at N' and N". With respect to MTX analogs, it was found that: (I) substitution of the glutamate side chain by z-aminoadipic acid. z-aminopimclic acid or /3aminoglutaric acid slightly affects its Ki: (2) a free z-carboxyl group on the amino acid side chain of MTX. or any free carboxyl group in that vicinity plays an important role in the inhibitory potency of MTX analogs to the enzyme; (3)esterilication or amidation ofthe z-carboxyl group of MTX decreases the inhibitory potency; and (4) free aspartyl or glutamyl conjugation through a peptide linkage to the ;~~boxyl group of the glutamate side chain decreases its K, to the enzyme by 5-and 8-fold respectively. Tetrahydrofolate analogs formed by inserting an ethylene, iminyl or a carbonyl bridge between the nitrogen at N" and N'" or by substitution at the NJ position were found to be poor inhibitors under our assay conditions.
Journal of Molecular Biology, 1996
Three steps along the pathway of binding, orientation of substrates and Department of Biochemistry and Biophysics, University of release of products are revealed by X-ray crystallographic structures of California, San Francisco ternary complexes of the wild-type Lactobacillus casei thymidylate synthase CA 94143-0448, USA enzyme. Each complex was formed by diffusion of either the cofactor 5,10-methylene-5,6,7,8-tetrahydrofolate or the folate analog 10-propargyl-5,8-dideazafolate into binary co-crystals of thymidylate synthase with 2'-deoxyuridine-5'-monophosphate. A two-substrate/enzyme complex is formed where the substrates remain unaltered. The imidazolidine ring is unopened and the pterin of the 5,10-methylene-5,6,7,8-tetrahydrofolate cofactor binds at an unproductive ''alternate'' site. We propose that the presence of the pterin at this site may represent an initial interaction with the enzyme that precedes all catalytic events. The structure of the 2'-deoxyuridine-5'-monophosphate and 10-propargyl-5,8-dideazafolate folate analog complex identifies both ligands in orientations favorable for the initiation of catalysis and resembles the productive complex. A product complex where the ligands have been converted into products of the thymidylate synthase reaction within the crystal, 2'-deoxythymidine-5'monophosphate and 7,8-dihydrofolate, shows how ligands are situated within the enzyme after catalysis and on the way to product release.
Concerted versus stepwise mechanism in thymidylate synthase
Journal of the American Chemical Society, 2014
Thymidylate synthase (TSase) catalyzes the intracellular de novo formation of thymidylate (a DNA building block) in most living organisms, making it a common target for chemotherapeutic and antibiotic drugs. Two mechanisms have been proposed for the rate-limiting hydride transfer step in TSase catalysis: a stepwise mechanism in which the hydride transfer precedes the cleavage of the covalent bond between the enzymatic cysteine and the product and a mechanism where both happen concertedly. Striking similarities between the enzyme-bound enolate intermediates formed in the initial and final step of the reaction supported the first mechanism, while QM/MM calculations favored the concerted mechanism. Here, we experimentally test these two possibilities using secondary kinetic isotope effect (KIE), mutagenesis study, and primary KIEs. The findings support the concerted mechanism and demonstrate the critical role of an active site arginine in substrate binding, activation of enzymatic nucl...