Catalysis by protein tyrosine phosphatases (PTPs) relies on the motion of a flexible protein loop (the WPD-loop) that carries a residue acting as a general acid/base catalyst during the PTP-catalyzed reaction. The orthogonal substitutions of a non-catalytic residue in the WPD-loops of YopH and PTP1B results in shifted pH-rate profiles, from an altered kinetic pKa of the nucleophilic cysteine. Compared to WT, the G352T YopH variant has a broadened pH-rate profile, similar activity at optimal pH, but significantly higher activity at low pH. Changes in the corresponding PTP1B T177G variant are more modest and in the opposite direction, with a narrowed pH profile and less activity in the most acidic range. Crystal structures of the variants show no structural perturbations, but suggest an increased preference for the WPD-loop closed conformation. Computational analysis confirms a shift in loop conformational equilibrium in favor of the closed conformation, arising from a combination of increased stability of the closed state and destabilization of the loop-open state. Simulations identify the origins of this population shift, revealing differences in the flexibility of the WPD-loop and neighboring regions. Our results demonstrate that changes to the pH dependency of catalysis by PTPs can result from small changes in amino acid composition in their WPD-loops affecting only loop dynamics and conformational equilibrium. The perturbation of kinetic pKa values of catalytic residues by non-chemical processes affords a means for nature to alter an enzyme’s pH dependency by a less disruptive path than altering electrostatic networks around catalytic residues themselves.

中文翻译：

---

WPD循环上的单个残基影响蛋白质酪氨酸磷酸酶中催化作用的pH依赖性。

蛋白质酪氨酸磷酸酶（PTP）的催化作用依赖于柔性蛋白质环（WPD环）的运动，该环带有在PTP催化反应期间充当一般酸/碱催化剂的残基。YopH和PTP1B的WPD环中非催化残基的正交取代导致亲核半胱氨酸的动力学pKa改变，导致pH速率分布发生变化。与WT相比，G352T YopH变体具有更宽的pH速率分布，在最佳pH下具有相似的活性，但在低pH下具有明显更高的活性。相应的PTP1B T177G变体的变化更适度，且方向相反，在最酸性范围内pH值变窄，活性降低。变体的晶体结构未显示结构扰动，但表明对WPD环闭合构象的偏好增加。计算分析证实，闭环构象平衡发生了变化，有利于闭环构象，这是由于闭环状态稳定性提高和开环状态不稳定导致的。模拟确定了这种人口转移的根源，揭示了WPD环路和邻近区域灵活性的差异。我们的结果表明，PTP催化pH依赖性的变化可能是由其WPD环中氨基酸组成的细微变化引起的，仅影响环动力学和构象平衡。非化学过程对催化残基的动力学pKa值的扰动为自然界提供了一种手段，可以通过比破坏催化残基自身周围的静电网络更少的破坏性途径来改变酶的pH依赖性。