Origin of the proton-transfer step in the cofactor-free (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase: effect of the basicity of an active site His residue.
Origin of the proton-transfer step in the cofactor-free (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase: effect of the basicity of an active site His residue.
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DOI：
10.1074/jbc.m113.543033
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发表时间：
2014-03-21
期刊：
影响因子：
--
通讯作者：
Scrutton NS
中科院分区：
文献类型：
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作者：
Hernandez-Ortega A;Quesne MG;Bui S;Heuts DP;Steiner RA;Heyes DJ;de Visser SP;Scrutton NS
背景：无辅酶双加氧酶的作用机制尚未明确。结果：(1H)-3-羟基-4-氧喹啉2,4-双加氧酶中His/Asp二联体突变强烈影响底物去质子化和整体催化。结论：揭示了His-251作为催化碱，Asp-126调节碱性的碱基机制。意义：许多双加氧酶通过去质子化激活其底物，这是随后与氧反应的必要步骤。双加氧酶催化多种化学反应，包括将氧结合到底物中，通常使用过渡金属或有机辅因子进行反应。细菌(1H)-3-羟基-4-氧喹啉2,4-双加氧酶（HOD）属于一类不需要任何辅助因子就能催化这种能量不利反应的加氧酶。在喹那丁代谢途径中，HOD分解其天然n -杂芳香底物的机制尚不完全清楚。采用实验和计算相结合的方法研究了催化循环的初始阶段。我们研究了活性位点His-251/Asp-126的作用，认为它参与了底物羟基去质子化，这是后续氧反应的关键要求。H251A和D126A突变体在稳态条件下的pH谱显示，pH值对其kcat和kcat/Km常数有很强的影响，在pH值为10.5时，kcat/Km分别下降了5500倍和9倍。在瞬态条件下的底物去质子化研究表明，这一步骤没有速率限制，WT HOD的pKa值为~ 7.2。发现了较大的溶剂同位素效应，pKa值在D2O中移至~ 8.3。晶体学和计算研究表明，突变对底物定位的影响很小。计算结果表明，His-251和Asp-126对初始反应的质子转移驱动力都是必不可少的。这项多学科研究明确支持了这样一种观点，即由His/Asp二元组合驱动的底物去质子化是其激活的必要条件。
Background: The mechanism of cofactor-free dioxygenases has not been clearly elucidated. Results: Mutation of the His/Asp dyad in (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase strongly affects substrate deprotonation and overall catalysis. Conclusion: Base mechanism is demonstrated where His-251 acts as catalytic base and Asp-126 modulates basicity. Significance: Many dioxygenases activate their substrates via deprotonation, which is an essential step for later reaction with oxygen. Dioxygenases catalyze a diverse range of chemical reactions that involve the incorporation of oxygen into a substrate and typically use a transition metal or organic cofactor for reaction. Bacterial (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) belongs to a class of oxygenases able to catalyze this energetically unfavorable reaction without any cofactor. In the quinaldine metabolic pathway, HOD breaks down its natural N-heteroaromatic substrate using a mechanism that is still incompletely understood. Experimental and computational approaches were combined to study the initial step of the catalytic cycle. We have investigated the role of the active site His-251/Asp-126 dyad, proposed to be involved in substrate hydroxyl group deprotonation, a critical requirement for subsequent oxygen reaction. The pH profiles obtained under steady-state conditions for the H251A and D126A variants show a strong pH effect on their kcat and kcat/Km constants, with a decrease in kcat/Km of 5500- and 9-fold at pH 10.5, respectively. Substrate deprotonation studies under transient-state conditions show that this step is not rate-limiting and yield a pKa value of ∼7.2 for WT HOD. A large solvent isotope effect was found, and the pKa value was shifted to ∼8.3 in D2O. Crystallographic and computational studies reveal that the mutations have a minor effect on substrate positioning. Computational work shows that both His-251 and Asp-126 are essential for the proton transfer driving force of the initial reaction. This multidisciplinary study offers unambiguous support to the view that substrate deprotonation, driven by the His/Asp dyad, is an essential requirement for its activation.