Morino Y, Yamasaki M, Tanase S, Nagashima F, Akasaka K, Imoto T, Miyazawa T
J Biol Chem. 1984 Mar 25;259(6):3877-82.
200 MHz proton nuclear magnetic resonance spectra were compared between the cytosolic (cAAT) and mitochondrial (mAAT) isoenzymes of aspartate aminotransferase (EC 2.6.1.1) from pig heart. The pattern of signal distribution in the whole spectral region differed considerably between the two isoenzymes, reflecting the difference in their amino acid sequences. A group of distinct signals were resolved at elevated temperatures (50 to 70 degrees C) in the low field region (9.0 to 7.5 ppm) of the spectra of both isoenzymes in the pyridoxal form. Most of these signals were also observable at 28 degrees C although some showed considerable line broadening. Among resonance lines in this spectral region, cAAT in the pyridoxal form showed four pH-titratable resonances with pKa of 9.54, 6.72, 5.69, and 4.87 at 28 degrees C. Variation in pK and line width of these signals indicated differences in the microenvironment of histidyl residues. On the other hand, mAAT showed six pH-titratable resonances with pKa of 6.73 (peak 2), 6.77 (peak 3), 6.07 (peak 4), 4.71 (peak 5), 4.54 (peak 6), and 4.33 (peak 7). Peaks 2, 3, and 4 were narrow and others were considerably broad. Thus, only part of the histidyl residues present in each isoenzyme (8 and 10 His/monomeric unit of cAAT and mAAT, respectively) appeared on the spectra as pH-titratable resonances. With both isoenzymes, chemical shift and pKa values of these signals obtained for the pyridoxal form were indistinguishable from those for the pyridoxamine form and the borohydride-reduced form. None of the observable signals were affected upon the interaction of cAAT with glutarate. By contrast, peaks 2 and 4 in mAAT showed subtle but distinct chemical shift changes upon complex formation with succinate, suggesting that these two resonances are due to histidyl residues located at the part of the enzyme molecule which undergoes a conformational change upon the interaction with the dicarboxylate.
对猪心脏天冬氨酸氨基转移酶（EC 2.6.1.1）的胞质同工酶（cAAT）和线粒体同工酶（mAAT）的200兆赫质子核磁共振谱进行了比较。在整个光谱区域中，两种同工酶的信号分布模式有很大差异，这反映了它们氨基酸序列的不同。在吡哆醛形式的两种同工酶光谱的低场区域（9.0至7.5 ppm），在升高的温度（50至70摄氏度）下分辨出一组不同的信号。尽管有些信号显示出明显的谱线展宽，但在28摄氏度时也能观察到这些信号中的大多数。在该光谱区域的共振线中，吡哆醛形式的cAAT在28摄氏度时显示出四个可进行pH滴定的共振峰，其pKa分别为9.54、6.72、5.69和4.87。这些信号的pK和线宽的变化表明组氨酸残基微环境的差异。另一方面，mAAT显示出六个可进行pH滴定的共振峰，其pKa分别为6.73（峰2）、6.77（峰3）、6.07（峰4）、4.71（峰5）、4.54（峰6）和4.33（峰7）。峰2、3和4很窄，其他峰则明显较宽。因此，每种同工酶中存在的组氨酸残基（cAAT和mAAT分别为每个单体单元8个和10个His）中只有一部分在光谱上表现为可进行pH滴定的共振峰。对于两种同工酶，吡哆醛形式获得的这些信号的化学位移和pKa值与吡哆胺形式和硼氢化物还原形式的化学位移和pKa值没有区别。cAAT与戊二酸相互作用时，没有一个可观察到的信号受到影响。相比之下，mAAT中的峰2和4在与琥珀酸形成复合物时显示出细微但明显的化学位移变化，这表明这两个共振峰是由于位于酶分子中与二羧酸相互作用时发生构象变化的部分的组氨酸残基引起的。
