Modulating the Nucleophile of a Glycoside Hydrolase through Site-Specific Incorporation of Fluoroglutamic Acids
Modulating the Nucleophile of a Glycoside Hydrolase through Site-Specific Incorporation of Fluoroglutamic Acids
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DOI：
10.1021/jacs.8b04235
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发表时间：
2018-07-04
影响因子：
15
通讯作者：
Withers, Stephen G.
中科院分区：
文献类型：
--
作者：
Kotzler, Miriam P.;Robinson, Kyle;Withers, Stephen G.
了解酶催化糖苷键水解的详细机制是非常重要的，不仅是设计定制的成本效益，稳定和具体的催化剂，而且开发特定的糖苷酶抑制剂作为治疗。保留糖基化酶在涉及共价糖基-酶中间体的双置换机制中使用两个关键的羧酸残基，通常是谷氨酸。一个Glu充当亲核试剂，而另一个充当一般的酸/碱。酶的能力的一个重要部分归因于这些关键残基提供的静电的“完美匹配”，这是一个非常难以在模型系统或酶本身中证明的假设。我们通过制备模型糖苷酶环状芽孢杆菌β-木聚糖酶（Bcx）的合成变体来实验性地探测这种协同作用，其中亲核体Glu 78被4-氟或4，4-二氟谷氨酸取代以逐渐降低亲核性。这些Bcx变体通过制备光学纯的氟代谷氨酸构建块、掺入合成肽并连接到Bcx的截短环状排列体上而半合成。通过测量改变的静电在活性位点上的酶的动力学常数的影响，我们表明，降低亲核体pKa由两个单位shits的pH值依赖的活性由一个pH值单位。使用不同离去基团能力的底物的线性自由能相关性表明，通过减少亲核催化，酶的协同机制被破坏并向解离途径转移。我们的研究代表了第一个将氟化谷氨酸位点特异性引入任何蛋白质的例子。此外，它提供了独特的见解亲核和酸/碱催化酶活性位点内的协同作用。
Understanding the detailed mechanisms of enzyme-catalyzed hydrolysis of the glycosidic bond is fundamentally important, not only to the design of tailored cost-efficient, stable and specific catalysts but also to the development of specific glycosidase inhibitors as therapeutics. Retaining glycosidases employ two key carboxylic acid residues, typically glutamic acids, in a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. One Glu functions as a nucleophile while the other acts as a general acid/base. A significant part of enzymatic proficiency is attributed to a "perfect match" of the electrostatics provided by these key residues, a hypothesis that has been remarkably difficult to prove in model systems or in enzymes themselves. We experimentally probe this synergy by preparing synthetic variants of a model glycosidase Bacillus circulans beta-xylanase (Bcx) with the nucleophile Glu78 substituted by 4-fluoro or 4,4-difluoroglutamic acid to progressively reduce nucleophilicity. These Bcx variants were semisynthesized by preparation of optically pure fluoroglutamic acid building blocks, incorporation into synthetic peptides, and ligation onto a truncated circular permutant of Bcx. By measuring the effect of altered electrostatics in the active site on enzyme kinetic constants, we show that lowering the nucleophile pKa by two units shits the pH-dependent activity by one pH unit. Linear free energy correlations using substrates of varying leaving group ability indicate that by reducing nucleophilic catalysis the concerted mechanism of the enzyme is disrupted and shifted toward a dissociative pathway. Our study represents the first example of site-specific introduction of fluorinated glutamic acids into any protein. Furthermore, it provides unique insights into the synergy of nucleophilic and acid/base catalysis within an enzyme active site.