Dzugaj A, Heyduk T, Buczyłko J, Kochman M
Arch Biochem Biophys. 1985 Jun;239(2):486-90. doi: 10.1016/0003-9861(85)90716-7.
At pH 6.3 both the native and subtilisin-digested fructose-1,6-bisphosphatase (Fru-P2-ase) molecules exhibit four fast-reacting thiol groups. The kinetic analysis shows that the pK value for the reaction of these thiols is 8.1. The increase of pH from 6.3 to 9.3 results in an uncovering of the remaining 20 thiol groups. In subtilisin-cleaved enzyme the rate of reaction of SH groups is considerably higher than in the native enzyme at pH 9.3, indicating changes in the microenvironments around thiols upon modification. A fluorescent label inserted on a fast-reacting SH group and neighboring NH2 group shifts the pH optimum of the enzyme to alkaline region and decreases its sensitivity toward AMP. Spectral analysis of labeled enzyme indicates that the labeled region of protein is more hydrophilic upon proteolytic digestion. It is concluded that a molecule of subtilisin-digested enzyme has a more relaxed structure than the native enzyme. The relaxation of the enzyme to a new conformation is reflected by urea addition, which mimics the effect of subtilisin digestion. Correlation of enzyme activity versus its sensitivity toward AMP (I 0.5), shows that at low concentrations of urea the active-site region at pH 6.3 is more affected than the region of AMP binding.
在pH 6.3时，天然的和经枯草杆菌蛋白酶消化的果糖-1,6-二磷酸酶（Fru-P2-ase）分子均表现出四个快速反应的巯基。动力学分析表明，这些巯基反应的pK值为8.1。pH从6.3升高到9.3会导致另外20个巯基暴露。在枯草杆菌蛋白酶切割的酶中，在pH 9.3时SH基团的反应速率比天然酶中的高得多，这表明修饰后巯基周围的微环境发生了变化。插入到一个快速反应的SH基团和相邻NH2基团上的荧光标记将酶的最适pH移至碱性区域，并降低了其对AMP的敏感性。标记酶的光谱分析表明，蛋白质的标记区域在蛋白水解消化后更具亲水性。得出的结论是，经枯草杆菌蛋白酶消化的酶分子结构比天然酶更松散。酶向新构象的松弛通过添加尿素得以体现，尿素模拟了枯草杆菌蛋白酶消化的效果。酶活性与其对AMP的敏感性（I 0.5）的相关性表明，在低浓度尿素下，pH 6.3时的活性位点区域比AMP结合区域受影响更大。
