Phosphorus-31 nuclear magnetic resonance study on cytoplasmic aspartate aminotransferase from pig heart. A reinvestigation.
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.
D000818:Animals; D001219:Aspartate Aminotransferases; D003593:Cytoplasm / Q000201:enzymology; D005583:Fourier Analysis; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D009206:Myocardium / Q000201:enzymology*; D010758:Phosphorus; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013552:Swine
D010758:Phosphorus; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases