The present study sought to identify the structural determinants of aspartic protease structural stability and activity at elevated pH. Various hypotheses have been published regarding the features responsible for the unusual alkaline structural stability of renin, however, few structure-function studies have verified these claims. Using pepsin as a model system, and renin as a template for functional and structural alkaline stability, a rational re-design of pepsin was undertaken to identify residues contributing to the alkaline instability of pepsin-like aspartic proteases in regards to both structure and function. We constructed 13 mutants based on this strategy. Among them, mutants D159 L and D60A led to an increase in activity at elevated pH levels (p ≤ 0.05) and E4V and H53F were shown to retain native-like structure at elevated pH (p ≤ 0.05). Previously suggested carboxyl groups Asp¹¹, Asp¹¹⁸, and Glu¹³ were individually shown not to be responsible for the structural instability or lack of activity at neutral pH in pepsin. The importance of the β-barrel to structural stability was highlighted as the majority of the stabilizing residues identified, and 39% of the weakly conserved residues in the N-terminal lobe, were located in β-sheet strands of the barrel. The results of the present study indicate that alkaline stabilization of pepsin will require reduction of electrostatic repulsions and an improved understanding of the role of the hydrogen bonding network of the characteristic β-barrel.

本研究试图确定在升高的 pH 值下天冬氨酸蛋白酶结构稳定性和活性的结构决定因素。关于导致肾素异常碱性结构稳定性的特征已经发表了各种假设，但是，很少有结构-功能研究证实了这些说法。使用胃蛋白酶作为模型系统，肾素作为功能和结构碱性稳定性的模板，对胃蛋白酶进行了合理的重新设计，以鉴定导致胃蛋白酶样天冬氨酸蛋白酶在结构和功能方面的碱性不稳定性的残基。我们基于此策略构建了 13 个突变体。其中，突变体 D159 L 和 D60A 在升高的 pH 水平下导致活性增加（p≤ 0.05) 并且 E4V 和 H53F 显示在升高的 pH 值下保留类似天然的结构 ( p ≤ 0.05)。先前建议的羧基 Asp ¹¹、Asp ¹¹⁸和 Glu ¹³单独显示不是胃蛋白酶在中性 pH 下结构不稳定或缺乏活性的原因。β-桶对结构稳定性的重要性得到强调，因为大多数已鉴定的稳定残基和 N 端叶中 39% 的弱保守残基位于桶的 β-折叠链中。本研究的结果表明，胃蛋白酶的碱性稳定化将需要减少静电排斥并更好地理解特征 β 桶的氢键网络的作用。