Kim Y T, Kwok F, Churchich J E
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
J Biol Chem. 1988 Sep 25;263(27):13712-7.
Physical interactions between pyridoxal kinase and aspartate aminotransferase were detected by means of emission anisotropy and affinity chromatography techniques. Binding of aspartate aminotransferase (apoenzymes) to pyridoxal kinase tagged with a fluorescent probe was detected by emission anisotropy measurements at pH 6.8 (150 mM KCl). Upon saturation of the kinase with the aminotransferase, the emission anisotropy increases 22%. The protein complex is characterized by a dissociation constant of 3 microM. Time-dependent emission anisotropy measurements conducted with the mixture 5-naphthylamine-1-sulfonic acid-kinase aspartate aminotransferase (apoenzyme), revealed the presence of two rotational correlation times of phi 1 = 36 and phi 2 = 62 ns. The longer correlation time is attributed to the stable protein complex. By immobilizing one enzyme (pyridoxal kinase) through interactions with pyridoxal-Sepharose, it was possible to demonstrate that aspartate aminotransferase releases pyridoxal kinase. A test of compartmentation of pyridoxal-5-phosphate within the protein complex using alkaline phosphatase as trapping agent, indicates that the cofactor generated by the catalytic action of the kinase is channeled to the apotransaminase. The main function of the stable complex formed by the kinase and the aminotransferase is to hinder the release of free pyridoxal-5-phosphate into the bulk solvent.
通过发射各向异性和亲和色谱技术检测了吡哆醛激酶与天冬氨酸氨基转移酶之间的物理相互作用。在pH 6.8（150 mM KCl）条件下，通过发射各向异性测量检测了天冬氨酸氨基转移酶（脱辅基酶）与标记有荧光探针的吡哆醛激酶的结合。当激酶被氨基转移酶饱和时，发射各向异性增加22%。该蛋白质复合物的解离常数为3 microM。对5-萘胺-1-磺酸-激酶-天冬氨酸氨基转移酶（脱辅基酶）混合物进行的时间依赖性发射各向异性测量显示，存在两个旋转相关时间，分别为phi 1 = 36 ns和phi 2 = 62 ns。较长的相关时间归因于稳定的蛋白质复合物。通过与吡哆醛-琼脂糖相互作用固定一种酶（吡哆醛激酶），可以证明天冬氨酸氨基转移酶会释放吡哆醛激酶。使用碱性磷酸酶作为捕获剂对蛋白质复合物中磷酸吡哆醛的区室化进行的测试表明，激酶催化作用产生的辅因子被导向脱辅基转氨酶。激酶和氨基转移酶形成的稳定复合物的主要功能是阻碍游离磷酸吡哆醛释放到大量溶剂中。
