Björk I, Ylinenjärvi K
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center.
Biochemistry. 1992 Sep 15;31(36):8597-602. doi: 10.1021/bi00151a029.
The Cys-71-Cys-81 disulfide bond of the cysteine proteinase inhibitor, chicken cystatin, was specifically reduced by thioredoxin or low concentrations of dithiothreitol. This cleavage, followed by S-carbamoylmethylation, induced a conformational change of the protein, as evidenced by changes in isoelectric point and circular dichroism spectra and by an increased susceptibility to digestion by nontarget proteinases. The proteinase binding ability and the immunological properties of the inhibitor, however, were not detectably altered, indicating that the conformational change was limited to the region around the disrupted bond. In contrast, reduction of both disulfide bonds of cystatin by higher concentrations of dithiothreitol and subsequent alkylation led to the slow conversion of the inhibitor into two forms lacking proteinase binding ability, indicative of more extensive conformational changes. Together, these results suggest that the less accessible Cys-95-Cys-115 disulfide bond of chicken cystatin, but not the more accessible Cys-71-Cys-81 bond, is of importance for maintaining the conformation of the inhibitor required for binding of target proteinases.
半胱氨酸蛋白酶抑制剂鸡半胱氨酸蛋白酶抑制剂的Cys-71-Cys-81二硫键被硫氧还蛋白或低浓度的二硫苏糖醇特异性还原。这种切割，随后进行S-氨甲酰甲基化，诱导了蛋白质的构象变化，这通过等电点和圆二色光谱的变化以及对非靶标蛋白酶消化的敏感性增加得到证明。然而，抑制剂的蛋白酶结合能力和免疫特性没有明显改变，表明构象变化仅限于被破坏键周围的区域。相比之下，用更高浓度的二硫苏糖醇还原半胱氨酸蛋白酶抑制剂的两个二硫键并随后进行烷基化，导致抑制剂缓慢转化为两种缺乏蛋白酶结合能力的形式，这表明构象变化更广泛。总之，这些结果表明，鸡半胱氨酸蛋白酶抑制剂中较难接近的Cys-95-Cys-115二硫键，而不是较易接近的Cys-71-Cys-81键，对于维持靶标蛋白酶结合所需的抑制剂构象很重要。
