哺乳類の酸化損傷塩基修復酵素NTH1とOGG1の基質特異性と作用機序 : 第12回公開シンポジウム : 活性酸素の分子病態学
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Reactive oxygen species generate structurally diverse base lesions in DNA. In E.coli cells, oxidative pyrimidine lesions are removed by Endo III and Endo VIII, whereas oxidative purine lesions by Fpg. In the present study, substrate specificities and reaction mechanisms of NTH1, a mammalian homologue of Endo III, and OGG1, a mammalian functional homologue of Fpg, were characterized using defined oligonucleotide substrates and the obtained results were compared to those of Endo III and Fpg. Mouse NTH1 (mNTH1) recognized not only urea (UR), thymine glycol (TG), 5,6-dihydrothymine (DHT), and 5-hydroxyuracil (HOU) derived from pyrimidine bases but also formamidopyrimidine (FAPY) derived from guanine. With both mNTH1 and human NTH1, the activity for FAPY was comparable to TG. Unlike Endo III, the activities of mNTH1 for these lesions were essentially independent of paired bases. Human OGG1 (hOGG1) recognized 7, 8-dihydro-8-oxoguanine (OG) and FAPY. hOGG1 excised OG in a paired base-dependent manner but paired base effects were not evident for FAPY. The difference in the activity for the most preferred OG : C and the least preferred OG : A was 20-fold, while that for the most preferred FAPY : C and the least preferred FAPY : A was only 2.3-fold. These results indicate that FAPY : C is a good substrate for both NTH1 and OGG1, suggesting participation of the two enzymes in repair of this lesion in mammalian cells. In contrast, Endo III and Endo VIII recognized FAPY : C very poorly relative to TG. Determination of enzymatic parameters revealed that catalytic rate constants (k_<cat>) of mNTH1 and hOGG1 were much lower than those of Endo III and Fpg. It seems that distinctive rate determining steps for the enzymatic reaction are responsible for the differential paired base effects observed for Endo III and mNTH1. For Endo III with high k_<cat>, the rate determining step is flip out of a damaged base, thereby making the activity sensitive to paired bases. In contrast, for mNTH1 with low k_<cat>, the rate determining step is subsequent N-glycosylase and/or AP lyase, thereby making the activity insensitive to paired bases.
- 2001-12-22
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