Dissecting the electrostatic interactions and pH-dependent activity of a family 11 glycosidase
Dissecting the electrostatic interactions and pH-dependent activity of a family 11 glycosidase
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DOI：
10.1021/bi0105429
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发表时间：
2001-08-28
期刊：
影响因子：
2.9
通讯作者：
McIntosh, LP
中科院分区：
文献类型：
--
作者：
Joshi, MD;Sidhu, G;McIntosh, LP
先前对来自环状芽孢杆菌的低分子量家族11木聚糖酶的研究表明，亲核试剂（Glu 78，pK（a）4.6）和酸/碱催化剂（Glu 172，pK（a）6.7）的电离状态产生其pH依赖性活性谱。检查的晶体结构的BCX显示，Glu 78和Glu 172是在非常相似的环境中，并包围了几个化学等价和高度保守的活性位点残基。因此，没有明显的原因表明它们的表观pKa值不同。为了解决这个问题，实施了诱变方法，以确定哪些特征建立了这两种催化羧酸的pKa值（通过C-13 NMR直接测量，并通过pH依赖性活性曲线间接测量）。几个BCX变体的分析表明，电离形式的Glu 78优先稳定的Glu 172的部分由两个有序的残基，即Tyr 69和Gln 127贡献的较强的氢键。此外，理论pKa计算表明，Glu 78具有比Glu 172更低的pKa值，这是由于更小的去溶剂化能和与蛋白质内的永久部分电荷和可电离基团更有利的背景相互作用。Glu 172的pKa值又由于来自位置78处的带负电荷的谷氨酸的静电排斥而升高。结果还表明，所有的保守的活性位点残基的行为协调一致，在建立的pKa值的Glu 78和Glu 172，没有特定的残基是单独比任何其他更重要。通常，贡献正电荷和氢键的残基用于降低Glu 78和Glu 172的pKa值。氢键降低pKa值的程度很大程度上取决于氢键的长度（较短的键更低的pKa值）和供体的化学性质（COOH > OH > CONH 2）。相反，相邻的羧基可以降低或提高催化谷氨酸的pKa值，这取决于参与相互作用的残基的电离常数的静电键。虽然BCX的最适pH可以通过突变活性位点内的相邻残基而从-1.1变为+0.6 pH单位，但活性通常由于重要的基态和/或过渡态相互作用的丧失而受损。这些结果表明，最佳pH值的酶可能是最好的工程通过战略氨基酸取代，在“核心”活性位点以外的位置，静电影响催化残基，而不干扰其直接的结构环境。
Previous studies of the low molecular mass family 11 xylanase from Bacillus circulans show that the ionization state of the nucleophile (GIu78, pK(a) 4.6) and the acid/base catalyst (Glu 172, pK(a) 6.7) gives rise to its pH-dependent activity profile. Inspection of the crystal structure of BCX reveals that Glu78 and Glu172 are in very similar environments and are surrounded by several chemically equivalent and highly conserved active site residues. Hence, there are no obvious reasons why their apparent pKa values are different. To address this question, a mutagenic approach was implemented to determine what features establish the pKa values (measured directly by C-13 NMR and indirectly by pH-dependent activity profiles) of these two catalytic carboxylic acids. Analysis of several BCX variants indicates that the ionized form of Glu78 is preferentially stabilized over that of Glu 172 in part by stronger hydrogen bonds contributed by two well-ordered residues, namely, Tyr69 and Gln127. In addition, theoretical pKa calculations show that Glu78 has a lower pKa value than Glu 172 due to a smaller desolvation energy and more favorable background interactions with permanent partial charges and ionizable groups within the protein. The pKa value of Glu172 is in turn elevated due to electrostatic repulsion from the negatively charged glutamate at position 78. The results also indicate that all of the conserved active site residues act concertedly in establishing the pKa values of Glu78 and Glu 172, with no particular residue being singly more important than any of the others. In general, residues that contribute positive charges and hydrogen bonds serve to lower the pKa values of Glu78 and Glu172. The degree to which a hydrogen bond lowers a pKa value is largely dependent on the length of the hydrogen bond (shorter bonds lower pKa values more) and the chemical nature of the donor (COOH > OH > CONH2). In contrast, neighboring carboxyl groups can either lower or raise the pKa values of the catalytic glutamic acids depending upon the electrostatic linkage of the ionization constants of the residues involved in the interaction. While the pH optimum of BCX can be shifted from -1.1 to +0.6 pH units by mutating neighboring residues within the active site, activity is usually compromised due to the loss of important ground and/or transition state interactions. These results suggest that the pH optima of an enzyme might be best engineered by making strategic amino acid substitutions, at positions outside of the "core" active site, that electrostatically influence catalytic residues without perturbing their immediate structural environment.