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<p>We characterized the catabolic pathway of 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP) in the liver of rainbow trout (a representative of lower vertebrates) and determined the specific levels of all involved enzymatic activities. We analyzed the time-course kinetics of enzymatic hydrolysis of 40&#xa0;µM 8-oxo-dGTP, 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-diphosphate (8-oxo-dGDP), and 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-monophosphate (8-oxo-dGMP) by an extract of rainbow trout liver at pH 8.5 and 5.5. Four different enzymatic activities were found to participate in decomposition of 8-oxo-dGTP to a final product 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG), i.e., 8-oxo-dGTP pyrophosphohydrolase (specific activity at pH 8.5 107 U/mg protein, inactive at pH 5.5), 8-oxo-dGTP monophosphohydrolase (105 U/mg protein at pH 8.5, 181 U/mg protein at pH 5.5), 8-oxo-dGDP monophosphohydrolase (56 U/mg protein at pH 8.5, 188 U/mg protein at pH 5.5), and 8-oxo-dGMP phosphohydrolase (178 U/mg protein at pH 8.5, 485 U/mg protein at pH 5.5). At pH 8.5, all four enzymatic activities appeared to be dependent on magnesium ions (5&#xa0;mM), as they were inhibited by 12.5&#xa0;mM divalent metal ion chelator, ethylenediaminetetraacetic acid (EDTA). However, EDTA did not inhibit any of the three enzymatic activities detected at pH 5.5. There are two possible explanations for this phenomenon. The chelating power of EDTA in an acidic environment may be too low to remove Mg<sup>2+</sup> ions from the active sites of enzymes, or these reactions are catalyzed by a completely different set of magnesium-independent enzymes that exhibit an acidic pH optimum characteristic of lysosomal enzymes. A thirty-kilo Dalton ultrafiltrate of the liver extract contained exclusively Mg<sup>2+</sup>-dependent 8-oxo-dGTP pyrophosphohydrolase (207 U/mg protein) and very low 8-oxo-dGDP monophosphohydrolase (5 U/mg protein) activities at pH 8.5. This indicates that trout 8-oxo-dGTP pyrophosphohydrolase has features that are closer to mammalian MTH1 (NUDT1) enzymes than bacterial MutT proteins, which efficiently decompose both 8-oxo-dGTP and 8-oxo-dGDP.</p>

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Enzymatic catabolism of the mutagenic DNA precursor, 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP), in rainbow trout liver

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  • 发表日期：

    2026年7月1日

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  • Karol Bialkowski,
  • Tomasz Gnatowski

摘要

We characterized the catabolic pathway of 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP) in the liver of rainbow trout (a representative of lower vertebrates) and determined the specific levels of all involved enzymatic activities. We analyzed the time-course kinetics of enzymatic hydrolysis of 40 µM 8-oxo-dGTP, 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-diphosphate (8-oxo-dGDP), and 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-monophosphate (8-oxo-dGMP) by an extract of rainbow trout liver at pH 8.5 and 5.5. Four different enzymatic activities were found to participate in decomposition of 8-oxo-dGTP to a final product 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG), i.e., 8-oxo-dGTP pyrophosphohydrolase (specific activity at pH 8.5 107 U/mg protein, inactive at pH 5.5), 8-oxo-dGTP monophosphohydrolase (105 U/mg protein at pH 8.5, 181 U/mg protein at pH 5.5), 8-oxo-dGDP monophosphohydrolase (56 U/mg protein at pH 8.5, 188 U/mg protein at pH 5.5), and 8-oxo-dGMP phosphohydrolase (178 U/mg protein at pH 8.5, 485 U/mg protein at pH 5.5). At pH 8.5, all four enzymatic activities appeared to be dependent on magnesium ions (5 mM), as they were inhibited by 12.5 mM divalent metal ion chelator, ethylenediaminetetraacetic acid (EDTA). However, EDTA did not inhibit any of the three enzymatic activities detected at pH 5.5. There are two possible explanations for this phenomenon. The chelating power of EDTA in an acidic environment may be too low to remove Mg2+ ions from the active sites of enzymes, or these reactions are catalyzed by a completely different set of magnesium-independent enzymes that exhibit an acidic pH optimum characteristic of lysosomal enzymes. A thirty-kilo Dalton ultrafiltrate of the liver extract contained exclusively Mg2+-dependent 8-oxo-dGTP pyrophosphohydrolase (207 U/mg protein) and very low 8-oxo-dGDP monophosphohydrolase (5 U/mg protein) activities at pH 8.5. This indicates that trout 8-oxo-dGTP pyrophosphohydrolase has features that are closer to mammalian MTH1 (NUDT1) enzymes than bacterial MutT proteins, which efficiently decompose both 8-oxo-dGTP and 8-oxo-dGDP.