Veltman O R, Vriend G, Berendsen H J, Van den Burg B, Venema G, Eijsink V G
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Biochemistry. 1998 Apr 14;37(15):5312-9. doi: 10.1021/bi9725879.
Thermostable thermolysin-like proteases (TLPs), such as the TLP of Bacillus stearothermophilus CU-21 (TLP-ste), bind calcium in one double (Ca1,2) and two single (Ca3, Ca4) calcium binding sites. The single sites are absent in thermolabile TLPs, suggesting that they are determinants of (variation in) TLP stability. Mutations in the Ca3 and Ca4 sites of TLP-ste indeed reduced thermal stability, but only mutations in the Ca3 site affected the calcium-dependence of stability. The predominant effect of the Ca3 site results from the fact that the Ca3 site is part of a region of TLP-ste, which unfolding is crucial for thermal inactivation. Thermal inactivation is not caused by the absence of calcium from the Ca3 site per se, but rather by unfolding of a region of TLP-ste for which stability depends on the occupancy of the Ca3 site. In accordance with this concept is the observation that the effects of mutations in the Ca3 site could be compensated by stabilizing mutations near this site. In addition, it was observed that the contribution of calcium binding to the Ca3 was substantially reduced in extremely stable TLP-ste variants containing multiple stabilizing mutations in the Ca3 region. Apparently, in these latter variants, unfolding of the Ca3 region contributes little to the overall process of thermal inactivation.
热稳定的嗜热菌蛋白酶样蛋白酶（TLPs），如嗜热脂肪芽孢杆菌CU-21的TLP（TLP-ste），在一个双钙结合位点（Ca1,2）和两个单钙结合位点（Ca3、Ca4）结合钙。热不稳定的TLPs中不存在单钙结合位点，这表明它们是TLP稳定性（变化）的决定因素。TLP-ste的Ca3和Ca4位点发生突变确实会降低热稳定性，但只有Ca3位点的突变会影响稳定性的钙依赖性。Ca3位点的主要作用源于这样一个事实，即Ca3位点是TLP-ste一个区域的一部分，该区域的解折叠对于热失活至关重要。热失活不是由Ca3位点本身缺乏钙引起的，而是由TLP-ste一个区域的解折叠引起的，该区域的稳定性取决于Ca3位点的占据情况。与此概念相符的是，观察到Ca3位点突变的影响可以通过该位点附近的稳定突变得到补偿。此外，还观察到，在Ca3区域含有多个稳定突变的极其稳定的TLP-ste变体中，钙与Ca3结合的贡献显著降低。显然，在这些后一种变体中，Ca3区域的解折叠对热失活的整体过程贡献很小。
