ENERGETICS OF FOLDING SUBTILISIN BPN'
ENERGETICS OF FOLDING SUBTILISIN BPN'
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DOI：
10.1021/bi00136a003
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发表时间：
1992-06-02
期刊：
影响因子：
2.9
通讯作者：
GALLAGHER, DT
中科院分区：
文献类型：
--
作者：
BRYAN, P;ALEXANDER, P;GALLAGHER, DT
枯草杆菌蛋白酶是单体蛋白质的一个不寻常的例子，它对折叠和展开具有很大的动力学障碍。在这里，我们首次记录了枯草杆菌蛋白酶成熟形式的体外折叠。枯草杆菌蛋白酶通过定点诱变被修饰为蛋白水解无活性，从而允许系统地检查折叠的障碍。首先，钙结合到高亲和力的钙A-位的热力学和动力学已被测量的微量热法和荧光光谱。结合是一个化学驱动的过程，结合常数（K（a））等于7 × 10（6）M-1。此外，从A位点去除钙的动力学屏障（23 kcal/mol）显著大于结合的标准自由能（9.3 kcal/mol）。钙从枯草杆菌蛋白酶解离的动力学（例如，过量EDTA）因此非常慢（t1/2 = 1.3小时，25 ℃）。第二，为了测量枯草杆菌蛋白酶折叠的动力学独立于钙结合，从蛋白质中删除高亲和力钙结合位点。在低离子强度（I = 0.01）下，该突变体的重折叠需要数天。折叠速率加快了近100倍的离子强度增加10倍，这表明活化的自由能的一部分可能是静电。在相对高的离子强度（I = 0.5）下，突变体枯草杆菌蛋白酶的重折叠在25 ℃下不到1小时内完成。我们建议，部分的静电贡献折叠枯草杆菌蛋白酶的活化自由能有关的蛋白质的高电荷区，包括弱离子结合位点（站点B）。带电荷的氨基酸聚结以形成该离子结合位点可以对折叠呈现静电屏障，除非电荷可以在折叠期间被接近时被屏蔽。折叠和未折叠状态之间的高动力学屏障可能是细胞外微生物蛋白酶防止自身降解的基本特征。由于这种动力学屏障也是折叠的障碍，所以原序列可以在体内起催化折叠反应的作用。
Subtilisin is an unusual example of a monomeric protein with a substantial kinetic barrier to folding and unfolding. Here we document for the first time the in vitro folding of the mature form of subtilisin. Subtilisin was modified by site-directed mutagenesis to be proteolytically inactive, allowing the impediments to folding to be systematically examined. First, the thermodynamics and kinetics of calcium binding to the high-affinity calcium A-site have been measured by microcalorimetry and fluorescence spectroscopy. Binding is an enthalpically driven process with an association constant (K(a)) equal to 7 X 10(6) M-1. Furthermore, the kinetic barrier to calcium removal from the A-site (23 kcal/mol) is substantially larger than the standard free energy of binding (9.3 kcal/mol). The kinetics of calcium dissociation from subtilisin (e.g., in excess EDTA) are accordingly very slow (t1/2 = 1.3 h at 25-degrees-C). Second, to measure the kinetics of subtilisin folding independent of calcium binding, the high-affinity calcium binding site was deleted from the protein. At low ionic strength (I = 0.01) refolding of this mutant requires several days. The folding rate is accelerated almost 100-fold by a 10-fold increase in ionic strength, indicating that part of the free energy of activation may be electrostatic. At relatively high ionic strength (I = 0.5) refolding of the mutant subtilisin is complete in less than 1 h at 25-degrees-C. We suggest that part of the electrostatic contribution to the activation free energy for folding subtilisin is related to the highly charged region of the protein comprising the weak ion binding site (site B). The coalescing of the charged amino acids to form this ion binding site may present an electrostatic barrier to folding unless the charges can be shielded as they are brought into proximity during folding. A high kinetic barrier between the folded and unfolded states may be an essential characteristic of extracellular, microbial proteases to prevent autodegradation. Since this kinetic barrier is also an impediment to folding, the pro sequence may function in vivo to catalyze the folding reaction.