Specific Derivatization of the Active Site Tyrosine in dUTPase Perturbs Ligand Binding to the Active Site

Vértessy, Beáta G.; Persson, Rebecca; Rosengren, Anna; Zeppezauer, Michael; Nyman, Per Olof

doi:10.1006/bbrc.1996.0226

Cited by 42 publications

(45 citation statements)

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“…Protein Concentration-Protein concentration was measured either by Bradford's assay ((38), using the protein determination kit from Sigma, and bovine serum albumin as standard) or spectrophotometrically using A 1 cm, 280 nm 0.1% ϭ 0.32, 0.26, or 0.52, for the D. melanogaster dUTPase constructs 1-159, 1-187, or E. coli dUTPase, respectively, as calculated from amino acid composition or determined experimentally (39).…”

Section: Methodsmentioning

confidence: 99%

“…1 Binding Affinity of D. melanogaster dUTPase toward dUDP and dUMP-The nonhydrolyzable character of dUDP by D. melanogaster dUTPase, as established in the previous section, identifies this nucleoside diphosphate as a close substrate analogue useful for detailed analysis of the active site in equilibrium complexation experiments. In previous studies from this laboratory, CD spectroscopy was found useful for exploring the interaction of dUTPase from E. coli with dUDP (30,33,39). Following these earlier observations, spectra were recorded for the D. melanogaster dUTPase enzyme, for dUDP, and for an equimolar mixture thereof ( Fig.…”

Section: Table I Mass Spectrometric Analysis Of a Total Tryptic Digesmentioning

confidence: 98%

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Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

Kovari

Barábas

Takács

et al. 2004

Journal of Biological Chemistry

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dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54°C, 23°C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg 2؉ binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.

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Section: Methodsmentioning

confidence: 99%

Section: Table I Mass Spectrometric Analysis Of a Total Tryptic Digesmentioning

confidence: 98%

Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

Kovari

Barábas

Takács

et al. 2004

Journal of Biological Chemistry

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“…In addition, the product dUMP is the precursor for dTTP biosynthesis. The homotrimer dUTPase enzymes have three active sites, each constituted by conserved sequence motifs (motif I-V) from all three subunits [14][15][16][17][18]. In mycobacterial genomes, a bifunctional dCTP deaminase:dUTPase is additionally encoded [19].…”

Section: Introductionmentioning

confidence: 99%

Cross-species inhibition of dUTPase via the Staphylococcal Stl protein perturbs dNTP pool and colony formation in Mycobacterium

et al. 2015

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“…In the crystal structure of the equine infectious anemia virus (EIAV) enzyme, a Sr 2ϩ ion bound to both the ␣-and ␤-phosphate groups of dUDP has been localized (7), but in a position that would sterically prevent nucleophilic attack by a hydrolytic water molecule. Nonetheless, the catalytic importance of the C-terminal residues and the dependence of dUTPase action on Mg 2ϩ have been amply demonstrated by studies of the enzyme in solution (9)(10)(11)(12). Because dUTPase has acquired increasing attention as a potential target enzyme in cancer and antiretroviral chemotherapy, determination of the structural basis of its catalytic action is important in the design of chemotherapeutic agents.…”

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confidence: 99%

“…In the E. coli enzyme the C-terminal region is ordered only on binding 2Ј-deoxyuridine with a 5Ј-triphosphate group in the presence of a divalent metal ion (9,10,14). Because ordering of the C-terminal region of the polypeptide chain has been shown to be required for catalytic action by the enzyme (9)(10)(11), an approach must be adopted for structural analysis that can be applied to macromolecules in solution.…”

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confidence: 99%

Catalytic and structural role of the metal ion in dUTP pyrophosphatase

Mustafi

Békési

Vértessy

et al. 2003

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

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The metal ion dependence of the catalytic activity of recombinant Escherichia coli dUTP pyrophosphatase (dUTPase), an essential enzyme preventing incorporation of uracil into DNA, has been investigated by steady-state kinetic, electron paramagnetic resonance, and electron nuclear double resonance methods. Values of k cat and kcat͞Km were 4.5 ؎ 0.1 s ؊1 and 0.49 ؎ 0.1 ؋ 10 6 M ؊1 ⅐s ؊1 in the absence of divalent metal ions, 14.7 ؎ 2.2 s ؊1 and 25.1 ؎ 7.4 ؋ 10 6 M ؊1 ⅐s ؊1 in the presence of Mg 2؉ or Mn 2؉ , and 24.2 ؎ 3.6 s ؊1 and 2.4 ؎ 0.7 ؋ 10 6 M ؊1 ⅐s ؊1 when supported by VO 2؉ or bis(acetylacetonato)oxovanadium(IV). Binding of VO 2؉ to the enzyme in the presence of dUDP, a nonhydrolyzable substrate analog, was specific and competitive with Mg 2؉ . Electron paramagnetic resonance spectra of the ternary enzyme-VO 2؉ -chelatedUDP complex revealed a pattern of 31 P superhyperfine coupling specifying two structurally equivalent phosphate groups equatorially coordinated to the VO 2؉ ion. Proton electron nuclear double resonance spectra revealed an equatorial acetylacetonate ligand, indicating that one of the organic ligands had been displaced. By molecular graphics modeling, we show that the diphosphate group of enzyme-bound dUDP is sterically accessible to a hemi-chelate form of VO 2؉ . We propose a similar location compatible with all kinetic and spectroscopic results to account for the reactivity of VO 2؉ and the VO 2؉ -chelate in dUTP hydrolysis. In this location the metal ion could promote an ordered conformation of the Cterminal fragment that is obligatory for catalysis but dynamically flexible in the free enzyme.C ontrary to ordinary expectation, the nucleic acid uracil can be incorporated into DNA in the cell because DNA polymerases do not distinguish between thymine and uracil. The most important cellular vehicle for hindering this process is the enzyme dUTP pyrophosphatase (dUTPase) (1, 2). This enzyme hydrolyzes dUTP into dUMP and pyrophosphate, thereby removing dUTP from the DNA biosynthetic pathway. Although several high to medium resolution x-ray studies of dUTPases from human (3), bacterial (4-6), and viral (7, 8) sources have been reported, there is no understanding of the structural basis of the catalytic mechanism. The C-terminal portion of each subunit of this trimeric enzyme is partly disordered in crystals and does not contribute to the electron density map. Furthermore, the binding site of Mg 2ϩ in the active site has not been identified. In the crystal structure of the equine infectious anemia virus (EIAV) enzyme, a Sr 2ϩ ion bound to both the ␣-and ␤-phosphate groups of dUDP has been localized (7), but in a position that would sterically prevent nucleophilic attack by a hydrolytic water molecule. Nonetheless, the catalytic importance of the C-terminal residues and the dependence of dUTPase action on Mg 2ϩ have been amply demonstrated by studies of the enzyme in solution (9-12). Because dUTPase has acquired increasing attention as a potential target enzyme in cancer and antiretroviral chemot...

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Specific Derivatization of the Active Site Tyrosine in dUTPase Perturbs Ligand Binding to the Active Site

Cited by 42 publications

References 2 publications

Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

Cross-species inhibition of dUTPase via the Staphylococcal Stl protein perturbs dNTP pool and colony formation in Mycobacterium

Catalytic and structural role of the metal ion in dUTP pyrophosphatase

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