Genov N, Filippi B, Dolashka P, Wilson K S, Betzel C
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia.
Int J Pept Protein Res. 1995 Apr;45(4):391-400. doi: 10.1111/j.1399-3011.1995.tb01054.x.
The stability towards thermal and chemical (guanidine hydrochloride, GnHCl) denaturation of six inhibited subtilases (mesentericopeptidase, subtilisins BPN', Carlsberg and DY, proteinase K and thermitase) has been investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroic and fluorescence spectroscopy. Experiments in the absence and presence of extraneous calcium in the concentration range 2 x 10(-3)-10(-1) M were performed. The presence of calcium in the weak calcium binding site changes the denaturation drastically. The heat- (or GnHCl-) induced unfolding curves obtained using CD spectroscopy show two independent transitions which seem not to have been resolved before. The presence of Ca2+ in the second (third in the case of thermitase) binding site increases the Tm values by 11-21 degrees C and the delta GD(H2O) values obtained from denaturation experiments in GnHCl by 6.7-7.2 kcal/mol when an extraneous Ca2+ concentration of 2 x 10(-2) M was used. One interpretation is that the initial step of denaturation in the presence of added calcium is the formation of a partially unfolded intermediate form, retaining a highly ordered structure with 60-85% of the alpha-helix structure of the native enzyme. This intermediate then unfolds at a temperature considerably higher than that of the same proteinases in the absence of added Ca2+. The free energy of stabilization of the intermediates is increased by 1.8-2.8 times in comparison with that for the unfolding reactions of the subtilases with empty Ca2/Ca3 binding sites. A second interpretation is that the two steps in the unfolding curves correspond to enzyme without and with calcium in the weak binding site. Fluorescence experiments confirm the mechanism involving the formation of intermediate states. The results are discussed in relation to the X-ray models of the six subtilases.
通过动力学和平衡研究，考察了六种被抑制的枯草杆菌蛋白酶（肠肽酶、枯草杆菌蛋白酶BPN'、卡尔斯伯格蛋白酶和DY、蛋白酶K和嗜热菌蛋白酶）对热变性和化学变性（盐酸胍，GnHCl）的稳定性。通过圆二色光谱和荧光光谱监测去折叠过程。在浓度范围为2×10⁻³ - 10⁻¹ M的有无外源钙存在的情况下进行了实验。弱钙结合位点中钙的存在极大地改变了变性情况。使用圆二色光谱获得的热（或GnHCl）诱导去折叠曲线显示出两个独立的转变，这似乎是之前未被解析的。当使用2×10⁻² M的外源Ca²⁺浓度时，第二个（嗜热菌蛋白酶情况下为第三个）结合位点中Ca²⁺的存在使Tm值增加了11 - 21℃，并且从GnHCl中的变性实验获得的ΔGD(H₂O)值增加了6.7 - 7.2 kcal/mol。一种解释是，在添加钙的情况下，变性的初始步骤是形成部分去折叠的中间形式，保留具有天然酶60 - 85%的α - 螺旋结构的高度有序结构。然后这种中间体在比没有添加Ca²⁺时相同蛋白酶的温度高得多的温度下去折叠。与具有空Ca²⁺/Ca³⁺结合位点的枯草杆菌蛋白酶的去折叠反应相比，中间体的稳定自由能增加了1.8 - 2.8倍。另一种解释是，去折叠曲线中的两个步骤分别对应于弱结合位点中无钙和有钙的酶。荧光实验证实了涉及中间态形成的机制。结合六种枯草杆菌蛋白酶的X射线模型对结果进行了讨论。
