Transition state of ADP-ribosylation of acetyllysine catalyzed by Archaeoglobus fulgidus Sir2 determined by kinetic isotope effects and computational approaches.
Transition state of ADP-ribosylation of acetyllysine catalyzed by Archaeoglobus fulgidus Sir2 determined by kinetic isotope effects and computational approaches.
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DOI：
10.1021/ja910342d
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发表时间：
2010-09-08
影响因子：
15
通讯作者：
Sauve AA
中科院分区：
文献类型：
--
作者：
Cen Y;Sauve AA
Sirtuins是分布在所有生命形式中的蛋白质修饰酶。这些酶结合NAD+（一种通用代谢物），并使其与乙酰赖氨酸残基反应以实现蛋白质侧链的脱乙酰化。这种NAD+依赖性的脱乙酰化反应已被观察到的sirtuin酶来自古细菌，真细菌，酵母，后生动物和哺乳动物物种，这表明保守的化学机制，这些酶。脱乙酰化的第一个化学步骤是NAD+与乙酰赖氨酸残基反应，形成酶结合的ADPR-肽酰亚胺中间体和烟酰胺。在这份手稿中，ADP-核糖基化乙酰赖氨酸的过渡态是解决了一个Escheaglobus fulgidus sirtuin（Af 2Sir 2）。用竞争底物法测得动力学同位素效应（KIE）分别为：[1 N-15 N] = 1.024（2），[1′N-14 C] = 1.014（4），[1′N-3 H] = 1.300（3），[2′N-3 H] =1.099（5），[4′N-3 H] = 0.997（2），[5′N-3 H] = 1.020（5），[4′N-18O] = 0.984（5）。使用计算方法（Gaussian 03和ISOEFF 98）计算候选过渡态结构的KIE，以匹配计算的和实验确定的KIE来求解过渡态。结果表明，该酶稳定了一个高度解离的oxocarbenium离子样过渡态，具有非常低的键级的离去基团烟酰胺和亲核试剂乙酰赖氨酸。一种协调但高度异步的取代机制形成了去乙酰化酶去乙酰化反应的ADPR-肽酰亚胺中间体。
Sirtuins are protein modifying enzymes distributed throughout all forms of life. These enzymes bind NAD+, a universal metabolite, and react it with acetyllysine residues to effect deacetylation of protein side chains. This NAD+-dependent deacetylation reaction has been observed for sirtuin enzymes derived from archaeal, eubacterial, yeast, metazoan and mammalian species, suggesting conserved chemical mechanisms for these enzymes. The first chemical step of deacetylation is the reaction of NAD+ with an acetyllysine residue which forms an enzyme-bound ADPR-peptidylimidate intermediate and nicotinamide. In this manuscript, the transition state for the ADP-ribosylation of acetyllysine is solved for an Archaeaglobus fulgidus sirtuin (Af2Sir2). Kinetic isotope effects (KIEs) were obtained by the competitive substrate method and were [1N-15N] = 1.024(2), [1′N-14C] = 1.014(4), [1′N-3H] = 1.300(3), [2′N-3H] =1.099(5), [4′N-3H] = 0.997(2), [5′N-3H] = 1.020(5), [4′N-18O] = 0.984(5). KIEs were calculated for candidate transition state structures using computational methods (Gaussian 03 and ISOEFF 98) in order to match computed and experimentally determined KIEs to solve the transition state. The results indicate that the enzyme stabilizes a highly dissociated oxocarbenium ion-like transition state with very low bond orders to the leaving group nicotinamide and the nucleophile acetyllysine. A concerted yet highly asynchronous substitution mechanism forms the ADPR-peptidylimidate intermediate of the sirtuin deacetylation reaction.
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影响因子：
9.8
作者：
Bordone L;Motta MC;Picard F;Robinson A;Jhala US;Apfeld J;McDonagh T;Lemieux M;McBurney M;Szilvasi A;Easlon EJ;Lin SJ;Guarente L
通讯作者：
Guarente L
影响因子：
16
作者：
Avalos, JL;Bever, KM;Wolberger, C
通讯作者：
Wolberger, C
影响因子：
11.4
作者：
García-Salcedo, JA;Gijón, P;Pays, E
通讯作者：
Pays, E
影响因子：
15
作者：
Greig, Ian R.;Macauley, Matthew S.;Vocadlo, David J.
通讯作者：
Vocadlo, David J.
影响因子：
16
作者：
Avalos, JL;Celic, I;Wolberger, C
通讯作者：
Wolberger, C