Illuminating the mechanistic roles of enzyme conformational dynamics
Illuminating the mechanistic roles of enzyme conformational dynamics
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DOI：
10.1073/pnas.0708600104
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发表时间：
2007-11-13
影响因子：
11.1
通讯作者：
Yang, Haw
中科院分区：
文献类型：
--
作者：
Hanson, Jeffrey A.;Duderstadt, Karl;Yang, Haw
许多酶塑造其结构以将底物封闭在其活性位点中，使得在每个催化循环期间可能需要构象重塑。在腺苷酸激酶（AK）中，这涉及酶的盖结构域的大幅度重排。使用我们的高分辨率单分子FRET的方法，我们直接遵循AK的结构域运动的催化时间尺度。为了定量测量酶的整个构象分布，我们应用了基于最大熵的方法来去除单分子数据中的光子计数噪声。该分析明确地表明，AK能够动态采样两种不同的状态，这与X射线晶体学观察到的状态有很好的相关性。出乎意料的是，平衡有利于封闭的，活性位点形成的配置，即使在没有基板。我们的实验进一步表明，与底物的相互作用，而不是锁定成一个紧凑的状态的酶，限制了空间的构象波动的程度和移动的酶的构象平衡向封闭的形式，通过增加盖子的关闭速率。将这些微观动力学整合到宏观动力学中，使我们能够将盖打开耦合的产物释放作为酶的限速步骤来建模。
Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme's lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK's domain movements on its catalytic time scale. To quantitatively measure the enzyme's entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly, the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme's conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme's rate-limiting step.