Koval Vladimir V, Kuznetsov Nikita A, Zharkov Dmitry O, Ishchenko Alexander A, Douglas Kenneth T, Nevinsky Georgy A, Fedorova Olga S
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Nucleic Acids Res. 2004 Feb 9;32(3):926-35. doi: 10.1093/nar/gkh237. Print 2004.
Formamidopyrimidine-DNA-glycosylase (Fpg protein, MutM) catalyses excision of 8-oxoguanine (8-oxoG) and other oxidatively damaged purines from DNA in a glycosylase/apurinic/apyrimidinic-lyase reaction. We report pre-steady-state kinetic analysis of Fpg action on oligonucleotide duplexes containing 8-oxo-2'-deoxyguanosine, natural abasic site or tetrahydrofuran (an uncleavable abasic site analogue). Monitoring Fpg intrinsic tryptophan fluorescence in stopped-flow experiments reveals multiple conformational transitions in the protein molecule during the catalytic cycle. At least four and five conformational transitions occur in Fpg during the interaction with abasic and 8-oxoG-containing substrates, respectively, within 2 ms to 10 s time range. These transitions reflect the stages of enzyme binding to DNA and lesion recognition with the mutual adjustment of DNA and enzyme structures to achieve catalytically competent conformation. Unlike these well-defined binding steps, catalytic stages are not associated with discernible fluorescence events. Only a single conformational change is detected for the cleavable substrates at times exceeding 10 s. The data obtained provide evidence that several fast sequential conformational changes occur in Fpg after binding to its substrate, converting the protein into a catalytically active conformation.
甲酰胺嘧啶 - DNA - 糖基化酶（Fpg蛋白，MutM）在糖基化酶/脱嘌呤/脱嘧啶裂合酶反应中催化从DNA切除8 - 氧代鸟嘌呤（8 - oxoG）和其他氧化损伤的嘌呤。我们报告了Fpg对含有8 - 氧代 - 2'- 脱氧鸟苷、天然无碱基位点或四氢呋喃（一种不可切割的无碱基位点类似物）的寡核苷酸双链体作用的稳态前动力学分析。在停流实验中监测Fpg内在色氨酸荧光揭示了催化循环中蛋白质分子的多个构象转变。在与无碱基和含8 - oxoG的底物相互作用期间，Fpg分别在2毫秒至10秒的时间范围内至少发生四个和五个构象转变。这些转变反映了酶与DNA结合以及损伤识别的阶段，同时DNA和酶结构相互调整以达到催化活性构象。与这些明确的结合步骤不同，催化阶段与可辨别的荧光事件无关。对于可切割底物，仅在超过10秒的时间检测到单一构象变化。获得的数据提供了证据，表明Fpg与底物结合后发生了几个快速连续的构象变化，将蛋白质转变为催化活性构象。
