Betzel C, Klupsch S, Branner S, Wilson K S
EMBL, Hamburg, Germany.
Adv Exp Med Biol. 1996;379:49-61. doi: 10.1007/978-1-4613-0319-0_7.
Savinase and Esperase (EC. 3.4.21.14) are secreted by the alkalophilic bacterium Bacillus lentus and are representatives of that subgroup of subtilisin enzymes with maximum stability in the range of pH 7 to 10 and high activity in the range of pH 8 to 12. The crystal structures of native Savinase and diisopropyl fluorophosphate (DFP) inhibited Esperase have been refined using X-ray data to 1.4 angstroms and 1.8 angstroms resolution respectively collected with synchrotron radiation. The structures were refined to R-factors (=(Sigma//Fo/-/Fc//)/(Sigma/Fo/)) of 16.4% for Esperase and 17.3% for Savinase. The sequence identity between the two enzymes is 66%. The structures are very similar to those of other Bacillus subtilisins. There are two calcium ions in each, equivalent to the strong and the weak sites in subtilisins Carlsberg and BPN'. The structures show novel features which can to some extent be related to their stability and activity. The large number of salt bridges in Esperase and Savinase is likely to contribute to the high thermal stability. Non-conservative substitutions and deletions in the hydrophobic binding pocket S1 as well as the more hydrophobic character of the substrate binding region probably contribute to the alkaline activity profile of the enzymes. Towards the end of the binding site there is an extra proline, Pro131, in Savinase near proline 129, forming a cluster that provides extra active-site rigidity compared with other subtilisins. On the other side of the active site of Esperase and Savinase, the tyrosine found in most other subtilisins is replaced by leucine and valine respectively. The tyrosine potentially interacts with substrate residue P6. At high pH, the negatively charged deprotonated tyrosine could interact unfavorably with the substrate, a possibility that is overcome by substitution with a neutral residue. This is probably one explanation for the shift of the activity profile of Esperase and Savinase to more alkaline pH.
嗜碱芽孢杆菌分泌的嗜热栖热芽孢杆菌蛋白酶（Savinase）和埃斯帕酶（Esperase，EC. 3.4.21.14）是枯草杆菌蛋白酶亚组的代表，在pH 7至10范围内具有最大稳定性，在pH 8至12范围内具有高活性。天然嗜热栖热芽孢杆菌蛋白酶和二异丙基氟磷酸酯（DFP）抑制的埃斯帕酶的晶体结构已分别使用X射线数据精修至1.4埃和1.8埃的分辨率，这些数据是通过同步辐射收集的。埃斯帕酶的结构精修至R因子（=(Sigma//Fo/-/Fc//)/(Sigma/Fo/)）为16.4%，嗜热栖热芽孢杆菌蛋白酶为17.3%。这两种酶之间的序列同一性为66%。其结构与其他枯草杆菌蛋白酶的结构非常相似。每种酶中有两个钙离子，相当于枯草杆菌蛋白酶卡尔伯格和BPN'中的强位点和弱位点。这些结构显示出一些新特征，在一定程度上与它们的稳定性和活性有关。埃斯帕酶和嗜热栖热芽孢杆菌蛋白酶中大量的盐桥可能有助于其高热稳定性。疏水结合口袋S1中的非保守取代和缺失以及底物结合区域更强的疏水性可能有助于这些酶的碱性活性谱。在结合位点末端附近，嗜热栖热芽孢杆菌蛋白酶中靠近脯氨酸129处有一个额外的脯氨酸Pro131，形成一个簇，与其他枯草杆菌蛋白酶相比，提供了额外的活性位点刚性。在埃斯帕酶和嗜热栖热芽孢杆菌蛋白酶活性位点的另一侧，大多数其他枯草杆菌蛋白酶中发现的酪氨酸分别被亮氨酸和缬氨酸取代。酪氨酸可能与底物残基P6相互作用。在高pH值下，带负电荷的去质子化酪氨酸可能与底物发生不利相互作用，而用中性残基取代可以克服这种可能性。这可能是埃斯帕酶和嗜热栖热芽孢杆菌蛋白酶活性谱向更碱性pH值转变的一种解释。
