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首页> 外文期刊>Biochemistry >Processivity, Substrate Positioning, and Binding: The Role of Polar Residues in a Family 18 Glycoside Hydrolase
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Processivity, Substrate Positioning, and Binding: The Role of Polar Residues in a Family 18 Glycoside Hydrolase

机译：持续性，基质位置和绑定：极性残基在家庭18糖苷水解酶中的作用。

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摘要

The enzymatic degradation of recalcitrant polysaccharides such as cellulose (beta-1,4-linked glucose) and chitin (beta-1,4-linked N-acetylglucosamine) by glycoside hydrolases (GHs) is of significant biological and economical importance. In nature, depolymerization is primarily accomplished by processive GHs, which remain attached to the substrate between subsequent hydrolytic reactions. Recent computational efforts have suggested that the processive ability of a GH is directly linked to the ligand binding free energy. The contribution of individual aromatic residues in the active site of these enzymes has been extensively studied. In this study, we offer the first experimental evidence confirming correlation of binding free energy and degree of processivity and evidence that polar residues are essential for maintaining processive ability. Exchanging Thr(276) with Ala in substrate binding subsite -2 in the processive ChiA of Serratia marcescens results in a decrease in both the enthalpy (2.6 and 3.8 kcal/mol) and free energy (0.5 and 2.2 kcal/mol) for the binding to the substrate (G1cNAc)(6) and the inhibitor allosamidin, respectively, compared to that of the wild type. Moreover, the initial apparent processivity as measured by [(G1cNAc)(2)]/[G1cNAc] ratios (17.1 +/- 0.4) and chitin degradation efficiency (20%) are greatly reduced for ChiA-T276A versus those of the wild type (30.1 +/- 1.5 and 75%, respectively). Mutation of Arg(172) to Ala reduces the level of recognition and positioning of the substrate into the active site. Molecular dynamics simulations indicate ChiA-R172A behaves like the wild type, but the dynamics of ChiA-T276A are greatly influenced by mutation, which is reflective of their influence on processivity.
机译：糖苷水解酶（GHs）对难降解多糖如纤维素（β-1,4-连接的葡萄糖）和几丁质（β-1,4-连接的N-乙酰氨基葡萄糖）的酶促降解具有重要的生物学和经济意义。实际上，解聚主要是通过连续的GH来完成的，在后续的水解反应之间，GH仍与底物相连。最近的计算努力表明，GH的加工能力直接与配体结合自由能相关。已经广泛研究了这些酶的活性位点中单个芳香族残基的贡献。在这项研究中，我们提供了第一个实验证据，证实了结合自由能与持续性的程度相关，并证明了极性残基对于维持持续性至关重要。在粘质沙雷氏菌的过程性ChiA中与底物结合亚位点-2中的Ala交换Thr（276）导致结合的焓（2.6和3.8 kcal / mol）和自由能（0.5和2.2 kcal / mol）均降低与野生型相比，分别对底物（G1cNAc）（6）和抑制剂异源异黄素的抑制作用更大。而且，与野生型相比，通过[（G1cNAc）（2）/ [G1cNAc]比（17.1 +/- 0.4）和几丁质降解效率（20％）测得的初始表观生产力大大降低了。 （分别为30.1 +/- 1.5和75％）。 Arg（172）突变为Ala会降低识别水平和将底物定位到活性位点中。分子动力学模拟表明ChiA-R172A的行为类似于野生型，但是ChiA-T276A的动力学受突变的影响很大，这反映了它们对合成能力的影响。

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