MECHANISM OF INACTIVATION OF HUMAN O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE BY O6-BENZYLGUANINE
MECHANISM OF INACTIVATION OF HUMAN O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE BY O6-BENZYLGUANINE
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DOI：
10.1021/bi00096a009
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发表时间：
1993-11-16
期刊：
影响因子：
2.9
通讯作者：
DOLAN, ME
中科院分区：
文献类型：
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作者：
PEGG, AE;BOOSALIS, M;DOLAN, ME
人o6 -烷基鸟嘌呤- dna烷基转移酶被低浓度的o6 -苄基鸟嘌呤迅速失活，但大肠杆菌ogt基因的烷基转移酶敏感性低得多，大肠杆菌ada基因的烷基转移酶和酵母的烷基转移酶不受影响。o6 -苄基-2′-脱氧鸟苷的效力低于碱，但仍然是人烷基转移酶的有效失活剂，对微生物蛋白没有影响。o6 -烯丙基鸟嘌呤的活性稍低，但在200 muM的条件下，在30分钟内仍能使人和Ogt的烷基转移酶完全失活，对Ada蛋白有轻微影响，对酵母的烷基转移酶没有影响。o4 -苄基胸腺嘧啶没有灭活任何烷基转移酶蛋白。o6 -苄基鸟嘌呤使人烷基转移酶失活，导致蛋白质中s -苄基半胱氨酸的形成和鸟嘌呤的化学计量生产。鸟嘌呤的生成速率遵循二级动力学（k = 600 M-1 s-1）。先前与甲基化DNA底物反应的烷基转移酶失活使其丧失了将o6 -苄基鸟嘌呤转化为鸟嘌呤的能力。这些结果表明，o6 -苄基鸟嘌呤通过作为烷基转移的底物和在蛋白质的受体位点形成s -苄基半胱氨酸而使蛋白质失活。o6 -苄基鸟嘌呤不能灭活微生物烷基转移酶可能是由于该位点的空间限制。与苯基衍生物相比，烯丙基的有效性降低与预期一致，其参与双分子位移反应的率较低，其灭活Ogt烷基转移酶的能力可能是由于其较小的尺寸允许进入该活性位点。这些研究明确表明，人烷基转移酶蛋白可以作用于缺乏多核苷酸结构的低分子量底物。除了用于灭活蛋白质外，这种底物也可用于哺乳动物烷基转移酶的测定。
Human O6-alkylguanine-DNA alkyltransferase was rapidly inactivated by low concentrations of O6-benzylguanine, but the alkyltransferase from the Escherichia coli ogt gene was much less sensitive and alkyltransferases from the E. coli ada gene or from yeast were not affected. O6-Benzyl-2'-deoxyguanosine was less potent than the base, but was still an effective inactivator of the human alkyltransferase and had no effect on the microbial proteins. O6-Allylguanine was somewhat less active, but still gave complete inactivation of both the human and Ogt alkyltransferases at 200 muM in 30 min, slightly affected the Ada protein, and had no effect on the yeast alkyltransferase. O4-Benzylthymidine did not inactivate any of the alkyltransferase proteins tested. Inactivation of the human alkyltransferase by O6-benzylguanine led to the formation of S-benzylcysteine in the protein and to the stoichiometric production of guanine. The rate of guanine formation followed second-order kinetics (k = 600 M-1 s-1). Prior inactivation of the alkyltransferase by reaction with a methylated DNA substrate abolished its ability to convert O6-benzylguanine into guanine. These results indicate that O6-benzylguanine inactivates the protein by acting as a substrate for alkyl transfer and by forming S-benzylcysteine at the acceptor site of the protein. The inability of O6-benzylguanine to inactivate the microbial alkyltransferases may be explained by steric constraints at this site. The reduced effectiveness of the allyl compared to the benzyl derivative is in accord with its expected, lower rate of participation in bimolecular displacement reactions, and its ability to inactivate the Ogt alkyltransferase may be explained by its smaller size permitting access to this active site. These studies unequivocally show that the human alkyltransferase protein can act on low molecular weight substrates lacking the polynucleotide structure. In addition to their use to inactivate the protein, such substrates may also prove useful for the assay of mammalian alkyltransferase.