Schnackerz K D
Biochim Biophys Acta. 1984 Sep 11;789(2):241-4. doi: 10.1016/0167-4838(84)90211-5.
Striking differences of the environment of the phosphate group of pyridoxal-P in cytoplasmic and mitochondrial aspartate aminotransferase have been reported. Since most details of the three-dimensional structure of the active sites of these isozymes are identical, it seemed difficult to rationalize the reported differences. Therefore, the cytoplasmic isozyme was reinvestigated using 31P-NMR at 72.86 MHz. The 31P chemical shift of the cofactor of this isozyme was found to be pH-dependent with a pKa of 6.2. In the presence of 100 mM succinate or 100 mM glutarate, the 31P chemical shift of bound pyridoxal-P remains at 4.71 or 4.79 ppm, respectively, in the pH range from 5.0 to 8.0, indicating that the phosphate group of the cofactor appears to be in its dianionic form. Reduction of the internal pyridoxal-P Schiff's base dramatically increases the pKa of the phosphate group of the phosphopyridoxyl moiety of the protein to 8.3. Hence, our results on the cytoplasmic isozyme are similar to those reported for the mitochondrial isozyme.
据报道，胞质和线粒体天冬氨酸转氨酶中磷酸吡哆醛的磷酸基团所处环境存在显著差异。由于这些同工酶活性位点的三维结构大部分细节是相同的，所以很难解释所报道的差异。因此，利用72.86兆赫的31P-NMR对胞质同工酶进行了重新研究。发现该同工酶辅因子的31P化学位移依赖于pH值，其pKa为6.2。在100毫摩尔琥珀酸盐或100毫摩尔戊二酸盐存在的情况下，结合的磷酸吡哆醛的31P化学位移在pH值5.0至8.0范围内分别保持在4.71或4.79 ppm，这表明辅因子的磷酸基团似乎处于其二价阴离子形式。内部磷酸吡哆醛席夫碱的还原显著提高了蛋白质磷酸吡哆醛部分磷酸基团的pKa至8.3。因此，我们关于胞质同工酶的结果与报道的线粒体同工酶的结果相似。
