MECHANISM OF 06-ALKYLGUANINE DNA ALKYLTRANSFERASE

O6-烷基鸟嘌呤-DNA烷基转移酶通过转移一种 从鸟嘌呤的O6位到蛋白质上的半胱氨酸的烷基链。 这种作用会产生原始的DNA，但酶的游离半胱氨酸不会 再生，酶活性被破坏。形成和形成了 持续的O6-甲基鸟嘌呤损伤被认为与 致癌。蛋白质影响甲基转移的机制 是未知的。 AGT的假定作用机制将在本实验中得到验证 建议如下。AGT通过以下方式激活半胱氨酸作为亲核剂 去质子化。鸟嘌呤通过质子化被增强为离开基团 异质原子的。半胱氨酸攻击甲基取代 鸟嘌呤在协同反应中。 将DNA中的甲基直接转移到半胱氨酸上 通过确定是否所有三个氢都被转移来进行调查 通过测定甲基的立体化学 调职。通过质子化将鸟嘌呤作为离开基团的活化 碱基上的氮将通过合成然后反应来探测 O6-甲基去氮鸟嘌呤底物类似于AGT。A的低反应性 特定O6-甲基去氮鸟嘌呤底物将意味着氮作为 质子接受者。确定AGT是否在离开时激活鸟嘌呤 通过核苷上6位上的氧的质子化来基团， S6-甲基-6-硫代鸟嘌呤和SE6-甲基-6-硒鸟嘌呤底物类似物 将被合成并与AGT反应。运动学分析 参数将指示蛋白质是否质子化氧 6个位置。将探讨半胱氨酸残基的质子化状态。 通过研究AGT与底物反应的pH曲线 和碘乙酰胺。可能存在的碱性氨基酸的同一性 AGT能使半胱氨酸去质子化的活性部位为 通过合成AGT然后与活性反应进行研究 现场定向烷基化剂。

O6-Alkylguanine-DNA alkyltransferase repairs damaged DNA by transferring an alkyl chain from the O6-position of guanine to a cysteine on the protein. This action produces the original DNA but the enzyme's free cysteine is not regenerated and the enzyme activity is destroyed. The formation and persistence of the O6-methylguanine lesion has been implicated in carcinogenesis. The mechanism by which the protein effects methyl transfer is not known. The postulated mechanism of action of AGT which will be tested in this proposal is as follows. AGT activates the cysteine as a nucleophile by deprotonation. The guanine is enhanced as a leaving group by protonation of a heteroatom. The cysteine attacks the methyl group displacing the guanine in a concerted reaction. The direct transfer of the methyl group from the DNA to the cysteine will be investigated by determining whether all three hydrogens are transferred with the methyl group and by determining the stereochemistry of the methyl transfer. The activation of guanine as a leaving group by protonation of a nitrogen on the nucleobase will be probed by synthesizing and then reacting O6-methyldeazaguanine substrate analogs with AGT. Low reactivity of a particular O6-methyldeazaguanine substrate will implicate that nitrogen as a proton acceptor. To determine if AGT activates guanine as a leaving group by protonation of the oxygen at the 6-position on the nucleoside, S6-methyl-6-thioguanine and Se6-methyl-6-selenoguanine substrate analogs will be synthesized and reacted with AGT. Analysis of the kinetic parameters will indicate whether the protein protonates the oxygen at the 6-position. The protonation state of the cysteine residue will be probed by investigating the pH profile of reactivity of AGT with the substrates and with iodoacetamide. The identity of possible basic amino acids in the active site of AGT which could deprotonate the cysteine will be investigated by synthesizing and then reacting AGT with active site-directed alkylating agents.