High-Speed Atomic Force Microscopy Reveals Factors Affecting the Processivity of Chitinases during Interfacial Enzymatic Hydrolysis of Crystalline Chitin
High-Speed Atomic Force Microscopy Reveals Factors Affecting the Processivity of Chitinases during Interfacial Enzymatic Hydrolysis of Crystalline Chitin
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高速原子力显微镜揭示了结晶甲壳素界面酶水解过程中影响几丁质酶持续合成能力的因素
DOI：
10.1021/acscatal.0c02751
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发表时间：
2020
期刊：
影响因子：
12.9
通讯作者：
Yang Qing
中科院分区：
文献类型：
--
作者：
Qu Mingbo;Watanabe-Nakayama Takahiro;Sun Shaopeng;Umeda Kenichi;Guo Xiaoxi;Liu Yuansheng;Ando Toshio;Yang Qing
结晶甲壳素的酶解是一种固-液界面催化过程，它高度依赖于酶的加工性，即在不释放底物的情况下，每个酶-底物络合事件中连续重复的催化循环次数。然而，影响酶的加工性的因素还没有完全被了解，因为在固-液界面中的加工性很难用生化方法确定。利用高速原子力显微镜(HS-AFM)，我们研究了昆虫几丁质酶鸡尾酒，其中含有一个外切几丁质酶(OfChi-h)和两个内切几丁质酶(OfChtI和OfChtII)，这两种酶是脱落旧角质层必不可少的酶。在单分子水平上，我们发现了内切酶是一个重要的因素，可以将Ofchi-h在结晶几丁质中的水解反应速度从25次提高到176次，显著增加了每几丁质酶浓度下新结合到甲壳素表面积单位上的Ofchi-h的数量，并将几丁质表面分子在几丁质表面的持续运行时间(1/koff)从0.48增加到3.13 S。在存在内切酶的情况下，Ofchi-h分子聚集在几丁质纤维的末端并形成簇，迅速破坏几丁质纤维。过程性引发包括初始的底物结合和络合，是影响过程性水解的一个因素。根据Ofchi-h分子在甲壳素原纤维上的排列行为，推测了甲壳素分子的顺向启动时间为0.22±0.07 S。这项工作提高了我们对结晶甲壳素水解液-固界面催化过程中外切几丁质酶协同作用的认识，突出了内切几丁质酶在促进过程中的作用。
Enzymatic hydrolysis of crystalline chitin is a solid–liquid interfacial catalysis process that is highly dependent on the enzymes’ processivity, namely, the number of catalytic cycles consecutively repeated per enzyme–substrate complexation event without the release of the substrate. However, the factors affecting an enzyme’s processivity are not fully understood, as processivity in a solid–liquid interface can be hardly determined biochemically. Using high-speed atomic force microscopy (HS-AFM), here we investigated an insect chitinase cocktail containing one exochitinase (OfChi-h) and two endochitinases (OfChtI and OfChtII), the indispensable enzymes for shedding old cuticle. At the single-molecule level, we have revealed endochitinases as a significant factor to elevate the processivity of OfChi-h from 25 to 176 reactions in crystalline chitin hydrolysis, to dramatically increase the number of OfChi-h newly bound onto a chitin surface area unit per chitinase concentration, and to increase the duration time of processive running OfChi-h molecules on the chitin surface (1/koff) from 0.48 to 3.13 s. In the presence of endochitinases, OfChi-h molecules were observed to crowd at the ends of chitin fibrils and form clusters that quickly ablate chitin fibrils. The processive initiation including initial OfChi-h-substrate binding and complexation is a factor that affects processive hydrolysis. The time for processive initiation was deduced to be 0.22 ± 0.07 s according to an aligning behavior of OfChi-h molecules on chitin fibrils. This work improves our knowledge of exo–endo chitinase synergism during the solid–liquid interfacial catalysis of crystalline chitin hydrolysis, highlighting the role of endochitinases in boosting processive hydrolysis.