Elucidation of the proton transport mechanism in human carbonic anhydrase II.
Elucidation of the proton transport mechanism in human carbonic anhydrase II.
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DOI：
10.1021/ja8091938
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发表时间：
2009-06-10
影响因子：
15
通讯作者：
Voth GA
中科院分区：
文献类型：
--
作者：
Maupin CM;McKenna R;Silverman DN;Voth GA
人碳酸酐酶II（HCA II）是已知最快的酶之一，其在其酶促反应中利用限速质子转运（PT）步骤。为了在原子水平上评估PT事件，在这项工作中使用了多态经验价键（MS-EVB）方法。据观察，在HCA II的PT事件利用一个短暂的活性位点水簇之间的催化锌结合的水/氢氧化物和质子穿梭残留物，His 64运输过量的质子。这个PT事件被发现是依赖于酶的能力，形成和稳定的活性位点的水簇，除了它的能力，以一个有利的构象定向His 64。对His 64不同取向的PT自由能垒的评价揭示了该残基作为质子转运体的重要作用，并阐明了其通过活性位点水对PT屏障的直接影响。有人建议，速率限制步骤之间振荡的活性位点水PT事件His 64和去/质子化的His 64取决于外源性缓冲液浓度和His 64的取向。在位置64处不存在PT受体/供体的情况下，发现过量质子将利用三种不同路径中的一种进入/离开活性位点。后者的结果不仅允许增加了解酶如何利用蛋白质/溶剂界面，以引导多余的质子到/从感兴趣的区域，它也提供了宝贵的洞察HCA II突变体的化学救援实验。
Human carbonic anhydrase II (HCA II) is one of the fastest known enzymes, which utilizes a rate-limiting proton transport (PT) step in its enzymatic reaction. To evaluate the PT event at an atomistic level, the multistate empirical valence bond (MS-EVB) method has been utilized in this work. It is observed that the PT event in HCA II exploits a transient active site water cluster to transport the excess proton between the catalytic zinc-bound water/hydroxide and the proton shuttling residue, His64. This PT event is found to be dependent on the enzyme's ability to form and stabilize the active site water cluster in addition to its ability to orient His64 in a favorable conformation. Evaluation of the PT free energy barrier for different orientations of His64 reveals this residue's vital role as a proton transporter and elucidates its direct effect on the barrier to PT through the active site water. It is suggested that the rate-limiting step oscillates between the active site water PT event to His64 and the de/protonation of His64 depending on the exogenous buffer concentration and the orientation of His64. In the absence of a PT acceptor/donor at position 64, it is found that the excess proton will utilize one of three distinct paths to enter/leave the active site. This latter result not only allows for an increased understanding of how enzymes capitalize on the protein/solvent interface to guide excess protons to/from areas of interest, it also provides valuable insight into the chemical rescue experiments on HCA II mutants.
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