1H NMR studies of aspartate aminotransferase. Histidyl residues of cytosolic and mitochondrial isoenzymes.
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.
D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008929:Mitochondria, Heart / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D011485:Protein Binding; D011730:Pyridoxal / Q000378:metabolism; D013552:Swine
D011730:Pyridoxal; D006639:Histidine; D001219:Aspartate Aminotransferases