Catalytic and structural role of the metal ion in dUTP pyrophosphatase

Devkumar Mustafi, Angela Bekesi, Beata G. Vertessy, Marvin W. Makinen

Research output: Article

35 Citations (Scopus)


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 kcat and kcat/Km were 4.5 ± 0.1 s-1 and 0.49 ± 0.1 × 106 M-1·s-1 in the absence of divalent metal ions, 14.7 ± 2.2 s-1 and 25.1 ± 7.4 × 106 M-1·s-1 in the presence of Mg2+ or Mn2+, and 24.2 ± 3.6 s-1 and 2.4 ± 0.7 × 106 M-1·s-1 when supported by VO2+ or bis(acetylacetonato)oxovanadium(IV). Binding of VO2+ to the enzyme in the presence of dUDP, a nonhydrolyzable substrate analog, was specific and competitive with Mg2+. Electron paramagnetic resonance spectra of the ternary enzyme-VO2+-chelate-dUDP complex revealed a pattern of 31P superhyperfine coupling specifying two structurally equivalent phosphate groups equatorially coordinated to the VO2+ 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 VO2+. We propose a similar location compatible with all kinetic and spectroscopic results to account for the reactivity of VO2+ and the VO2+-chelate in dUTP hydrolysis. In this location the metal ion could promote an ordered conformation of the C-terminal fragment that is obligatory for catalysis but dynamically flexible in the free enzyme.

Original languageEnglish
Pages (from-to)5670-5675
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number10
Publication statusPublished - máj. 13 2003

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