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首页> 外文期刊>Journal of Molecular Biology >Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase.
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Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase.

机译：氢键合和催化：关于单个氨基酸取代如何改变糖苷酶最适pH值的新颖解释。

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摘要

The pH optima of family 11 xylanases are well correlated with the nature of the residue adjacent to the acid/base catalyst. In xylanases that function optimally under acidic conditions, this residue is aspartic acid, whereas it is asparagine in those that function under more alkaline conditions. Previous studies of wild-type (WT) Bacillus circulans xylanase (BCX), with an asparagine residue at position 35, demonstrated that its pH-dependent activity follows the ionization states of the nucleophile Glu78 (pKa 4.6) and the acid/base catalyst Glu172 (pKa 6.7). As predicted from sequence comparisons, substitution of this asparagine residue with an aspartic acid residue (N35D BCX) shifts its pH optimum from 5.7 to 4.6, with an approximately 20% increase in activity. The bell-shaped pH-activity profile of this mutant enzyme follows apparent pKa values of 3.5 and 5.8. Based on 13C-NMR titrations, the predominant pKa values of its active-site carboxyl groups are 3.7 (Asp35), 5.7 (Glu78) and 8.4 (Glu172). Thus, in contrast to the WT enzyme, the pH-activity profile of N35D BCX appears to be set by Asp35 and Glu78. Mutational, kinetic, and structural studies of N35D BCX, both in its native and covalently modified 2-fluoro-xylobiosyl glycosyl-enzyme intermediate states, reveal that the xylanase still follows a double-displacement mechanism with Glu78 serving as the nucleophile. We therefore propose that Asp35 and Glu172 function together as the general acid/base catalyst, and that N35D BCX exhibits a "reverse protonation" mechanism in which it is catalytically active when Asp35, with the lower pKa, is protonated, while Glu78, with the higher pKa, is deprotonated. This implies that the mutant enzyme must have an inherent catalytic efficiency at least 100-fold higher than that of the parental WT, because only approximately 1% of its population is in the correct ionization state for catalysis at its pH optimum. The increased efficiency of N35D BCX, and by inference all "acidic" family 11 xylanases, is attributed to the formation of a short (2.7 A) hydrogen bond between Asp35 and Glu172, observed in the crystal structure of the glycosyl-enzyme intermediate of this enzyme, that will substantially stabilize the transition state for glycosyl transfer. Such a mechanism may be much more commonly employed than is generally realized, necessitating careful analysis of the pH-dependence of enzymatic catalysis.
机译：11族木聚糖酶的最适pH与与酸/碱催化剂相邻的残基的性质良好相关。在酸性条件下发挥最佳功能的木聚糖酶中，该残基是天冬氨酸，而在碱性条件下发挥作用的那些是天冬酰胺。先前对野生型（WT）芽孢杆菌木聚糖酶（BCX）的研究在位置35处有天冬酰胺残基，表明其pH依赖性活性遵循亲核试剂Glu78（pKa 4.6）和酸/碱催化剂Glu172的电离状态。 （pKa 6.7）。正如序列比较所预测的那样，用天冬氨酸残基（N35D BCX）取代该天冬酰胺残基会将其最佳pH值从5.7变为4.6，活性增加约20％。该突变酶的钟形pH-活性曲线遵循3.5和5.8的明显pKa值。基于13 C-NMR滴定，其活性部位羧基的主要pKa值为3.7（Asp35），5.7（Glu78）和8.4（Glu172）。因此，与WT酶相反，N35D BCX的pH活性曲线似乎由Asp35和Glu78设定。 N35D BCX在其天然和共价修饰的2-氟-木糖基糖基酶中间状态下的突变，动力学和结构研究表明，木聚糖酶仍遵循双排位机制，其中Glu78充当亲核试剂。因此，我们建议Asp35和Glu172共同充当一般的酸/碱催化剂，并且N35D BCX表现出“逆质子化”机制，其中当具有较低pKa的Asp35被质子化时，它具有催化活性，而具有低pKa的Asp35被质子化。较高的pKa被去质子化。这意味着突变酶必须具有比亲代WT高至少100倍的固有催化效率，因为只有大约1％的突变体处于正确的电离状态才能在最适pH下进行催化。 N35D BCX效率的提高以及通过推断所有“酸性”家族11木聚糖酶的原因是，在该糖基酶中间产物的晶体结构中观察到Asp35和Glu172之间形成了一个短的（2.7 A）氢键。酶，将基本稳定糖基转移的过渡态。这种机制可能比通常所采用的机制更为普遍，因此需要仔细分析酶催化的pH依赖性。

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