Catalytic and structural role of the metal ion in dUTP pyrophosphatase

Abstract

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...

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

“…Enzyme and nucleotide stock solutions were incubated with Chelex resin for 30 min, on ice, followed by pelleting the resin in an Eppendorf centrifuge. Chelex-treated components were shown to contain less than 0.1 ppm of magnesium or manganese using atomic absorption spectroscopy on a Varian Spectra AA220 spectrophotometer, as described previously (42).…”

“…Enzymes involved in the metabolism of nucleotide phosphates and DNA or RNA usually require divalent metal ions as co-factors. Recently, it was shown that dUTPase from E. coli also possesses a similar catalytic activity in the absence of Mg 2ϩ (42). It seems that dUTPases in general might have a less stringent need for Mg 2ϩ , which is therefore not an obligate but an enhancer co-factor of these enzymes.…”

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

“…Protons released in the dUTPase reaction were detected by phenol red pH indicator in 1 mM HEPES pH 7.5 buffer also containing 150 mM KCl, 40 µM phenol red (Merck) and 5 mM MgCl 2 [34][35][36][37] …”

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

“…1 An additional enzymatic activity, deamination of dCTP (deoxycytidine triphosphate) to dUTP, may also be carried out by bifunctional dUTPases in Archaea [2][3][4][5] and also in Mycobacterium tuberculosis (M. tuberculosis), which also has a monofunctional dUTPase. 6 The dUTPases comprise two structurally different classes: 7 first, the β sheet−type dUTPases, possessed by most organisms, employ a one-metal ion catalytic mechanism, [8][9] and usually form a homotrimer 10 or a monomer, 11 while the second class, the α helical-type dUTPases, possessed by kinetoplastids, 12 form dimers and employ a two-metal ion catalytic mechanism. [13][14] In β sheet-type trimeric dUTPases, each active site is constituted from motifs of all three protomers, which are all required to interact for achieving proper enzymatic activity.…”