Abstract
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.
Highlights
200 MHz proton nuclear magnetic resonance spectra zymic forms in mammalian tissues: one is localized in mitowere compared between the cytosolicand mi- chondria and the other in cytosol [1,2,3]
For this reason, NMR studies on this of the histidyl residues present in each isoenzyme (8 enzyme have been confined to the observation on the coenand 10 His/monomeric unit ofcAAT and mAAT, re- zyme phosphorus [22, 23] and on the ligand molecules interspectively) appeared on the spectra as pH-titratable acting with the enzyme [24,25,26,27,28,29]
None of the observable signals wereaffected upon the interaction of cAATwith glutarate.By contrast, peaks 2 and 4 in mAAT showed subtle but distinctchemical shift changes upon complexformation with succinate, suggesting that these tworesonances aredue to histidyl residues located at the part of the enzymemolecule proton NMR spectrum
Summary
Yoshimasa Morino,Masaki Yamasaki, Sumio Tanase, Fujio Nagashima, Kazuyuki AkasakaS, Toshiaki JmotoSP, and Tatsuo Miyazawall. 8 histidyl residues/monomeric unit of the enzyme [6], only ppm five resonance lines were observed in the spectral region where histidyl C-2 proton resonates Four of these distinct signals exhibited pH-dependent chemical shift changes (Fig. 3). The temperature-dependent upfield shift of peaks 3 and 6 (Fig. 2A) may be due to the lowering in pK values [43] for these histidyl C-2 protons as observedwithsome histidyl residues in pancreatic ribonuclease [44]. The spectrum differed considerably from that of cAAT (see Fig. l ) , reflecting the difference in the primary structure between these two isoenzymes.The effect of temperature on the proton NMR spectra of mAAT was studied as in the case of cAAT.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
