Granon S, Kerfelec B, Chapus C
Centre de Biochimie et de Biologie Moléculaire du Centre National de la Recherche Scientifique, Marseille, France.
J Biol Chem. 1990 Jun 25;265(18):10383-8.
A spectrofluorimetric investigation of the interactions between the subunits of the pancreatic bovine procarboxypeptidase A ternary complex was carried out after covalent insertion of a fluorescent probe at the active center of one of the constituent subunits. The specific insertion of an anthraniloyl group at the active center of subunit II free or bound to subunit I, after its conversion into chymotrypsin II, allowed us to determine the value of the dissociation constant between subunit I and anthraniloyl-chymotrypsin II (Kd = 0.7 +/- 0.1 x 10(-7) M) and between subunit III and the binary complex subunit I-anthraniloyl-chymotrypsin II (Kd = 1.6 +/- 0.3 x 10(-7) M). Moreover, the influence of the association on the flexibility of the active center of chymotrypsin II was deduced from fluorescence polarization measurements and rotational correlation time determination of anthraniloyl-chymotrypsin II free or bound to subunit I. The anthraniloyl group has no motion independently of the whole chymotrypsin II molecule and the binding of subunit I to anthraniloyl-chymotrypsin II results in an increase of the rigidity of the active site in the latter protein.
在将荧光探针共价插入胰腺牛羧肽酶A三元复合物的一个组成亚基的活性中心后，对其亚基间的相互作用进行了荧光光谱研究。在将亚基II转化为胰凝乳蛋白酶II后，将邻氨基苯甲酰基特异性插入游离的或与亚基I结合的亚基II的活性中心，这使我们能够确定亚基I与邻氨基苯甲酰基 - 胰凝乳蛋白酶II之间的解离常数（Kd = 0.7 +/- 0.1 x 10(-7) M）以及亚基III与二元复合物亚基I - 邻氨基苯甲酰基 - 胰凝乳蛋白酶II之间的解离常数（Kd = 1.6 +/- 0.3 x 10(-7) M）。此外，通过对游离的或与亚基I结合的邻氨基苯甲酰基 - 胰凝乳蛋白酶II进行荧光偏振测量和旋转相关时间测定，推断出这种结合对胰凝乳蛋白酶II活性中心灵活性的影响。邻氨基苯甲酰基不能独立于整个胰凝乳蛋白酶II分子运动，亚基I与邻氨基苯甲酰基 - 胰凝乳蛋白酶II的结合导致后者蛋白质活性位点刚性增加。
