Wohlfahrt Gerd, Pellikka Tarmo, Boer Harry, Teeri Tuula T, Koivula Anu
Orion Pharma, P.O. Box 65, FIN-02101 Espoo, Finland.
Biochemistry. 2003 Sep 2;42(34):10095-103. doi: 10.1021/bi034954o.
Two carboxylic acid side chains can, depending on their geometry and environment, share a proton in a hydrogen bond and form a carboxyl-carboxylate pair. In the Trichoderma reesei cellobiohydrolase Cel6A structure, five carboxyl-carboxylate pairs are observed. One of these pairs (D175-D221) is involved in catalysis, and three other pairs are found in, or close to the two surface loops covering the active site tunnel of the catalytic domain. To stabilize Cel6A at alkaline pH values, where deprotonation of the carboxylic acids leads to repulsion of their side chains, we designed two mutant enzymes. In the first mutant, one carboxyl-carboxylate pair (E107-E399) was replaced by a corresponding amide-carboxylate pair (Q107-E399), and in the second mutant, all three carboxyl-carboxylate pairs (E107-E399, D170-E184, and D366-D419) were mutated in a similar manner. The unfolding studies using both intrinsic tryptophan fluorescence and far-ultraviolet circular dichroism spectroscopy at different pH values demonstrate that the unfolding temperature (T(m)) of both mutants has changed, resulting in destabilization of the mutant enzymes at acidic pH and stabilization at alkaline pH. The effect of stabilization seems additive, as a Cel6A triple mutant is the most stable enzyme variant. This increased stability is also reflected in the 2- or 4-fold increased half-life of the two mutants at alkaline pH, while the catalytic rate on cellotetraose (at t = 0) has not changed. Increased operational stability at alkaline pH was also observed on insoluble cellulosic substrates. Local conformational changes are suggested to take place in the active site loops of Cel6A wild-type enzyme at elevated pHs (pH 7), affecting to the end-product spectrum on insoluble cellulose. The triple mutant does not show such pH-dependent behavior. Overall, our results demonstrate that carboxyl-carboxylate pair engineering is a useful tool to alter pH-dependent protein behavior.
根据其几何形状和环境，两个羧酸侧链可以在氢键中共享一个质子，形成羧基 - 羧酸盐对。在里氏木霉纤维二糖水解酶Cel6A的结构中，观察到五个羧基 - 羧酸盐对。其中一对（D175 - D221）参与催化，另外三对位于覆盖催化结构域活性位点通道的两个表面环中或其附近。为了在碱性pH值下稳定Cel6A，此时羧酸的去质子化会导致其侧链相互排斥，我们设计了两种突变酶。在第一个突变体中，一对羧基 - 羧酸盐对（E107 - E399）被相应的酰胺 - 羧酸盐对（Q107 - E399）取代，在第二个突变体中，所有三对羧基 - 羧酸盐对（E107 - E399、D170 - E184和D366 - D419）以类似方式发生突变。使用内在色氨酸荧光和远紫外圆二色光谱在不同pH值下进行的去折叠研究表明，两个突变体的去折叠温度（T(m)）都发生了变化，导致突变酶在酸性pH下不稳定，而在碱性pH下稳定。稳定作用似乎具有加和性，因为Cel6A三重突变体是最稳定的酶变体。这种增加的稳定性还体现在两个突变体在碱性pH下的半衰期增加了2倍或4倍，而对纤维四糖的催化速率（在t = 0时）没有变化。在不溶性纤维素底物上也观察到在碱性pH下操作稳定性增加。有人提出，在较高pH值（pH 7）下，Cel6A野生型酶的活性位点环会发生局部构象变化，这会影响不溶性纤维素上的终产物谱。三重突变体没有表现出这种pH依赖性行为。总体而言，我们的结果表明，羧基 - 羧酸盐对工程是改变pH依赖性蛋白质行为的有用工具。
