固定化酶的结构和功能异质性的相关性,ACS Nano

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固定化酶的结构和功能异质性的相关性
ACS Nano ( IF 15.8 ) Pub Date : 2018-08-01 00:00:00 , DOI: 10.1021/acsnano.8b02956
Daniel F. Kienle ₁ , Rebecca M. Falatach ₁ , Joel L. Kaar ₁ , Daniel K. Schwartz ₁

Affiliation

许多纳米生物技术的应用依赖于功能性生物大分子与合成纳米材料的稳定有效结合。不幸的是，由于难以区分不同的分子水平机制，固定酶普遍丧失活性的原因仍然知之甚少。在这里，我们采用互补的单分子荧光的方法独立地测量固定化的在结构和功能（影响我。Ë（，NfsB）的底物结合动力学）。随机统计建模方法用于明确量化固定化NfsB结构动力学对功能的影响，使我们能够明确区分由于构象和可及性引起的影响。有趣的是，我们发现与位点特异性的NfsB相比，非特异性的NfsB表现出更高的稳定性。但是，位点特异性束缚的NfsB的折叠状态具有更快的底物结合率，表明活性位点可及性得到改善。这证明了与竞争性生物缀合方法相关的意料之外的内在取舍，表明可能有必要在构象稳定性与活动站点可访问性。这种对固定化影响的细致入微的观点将有助于酶与合成纳米材料整合的合理方法。

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Correlating Structural and Functional Heterogeneity of Immobilized Enzymes

Many nanobiotechnology applications rely on stable and efficient integration of functional biomacromolecules with synthetic nanomaterials. Unfortunately, the reasons for the ubiquitous loss of activity of immobilized enzymes remain poorly understood due to the difficulty in distinguishing between distinct molecular-level mechanisms. Here, we employ complementary single-molecule fluorescence methods that independently measure the impact of immobilization on the structure and function (i.e., substrate binding kinetics) of nitroreductase (NfsB). Stochastic statistical modeling methods were used to unambiguously quantify the effects of immobilized NfsB structural dynamics on function, allowing us to explicitly separate effects due to conformation and accessibility. Interestingly, we found that nonspecifically tethered NfsB exhibited enhanced stability compared to site-specifically tethered NfsB; however, the folded state of site-specifically tethered NfsB had faster substrate binding rates, suggesting improved active site accessibility. This demonstrated an unexpected intrinsic trade-off associated with competing bioconjugation methods, suggesting that it may be necessary to balance conformational stability versus active site accessibility. This nuanced view of the impact of immobilization will facilitate a rational approach to the integration of enzymes with synthetic nanomaterials.

更新日期：2018-08-01

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