title: 31P nuclear-magnetic-resonance studies of pyridoxal and pyridoxamine phosphates.  Interaction with cytoplasmic aspartate transaminase.
abstract: The 31P nuclear magnetic resonance (NMR) spectrum of the phosphate in free pyridoxal or pyridoxamine phosphate reveals a resonance signal that is coupled to the methylene protons of the 5-CH2 with JHP of 6.0 +/- 0.3 Hz. Proton noise decoupling results in a single signal with a pH-dependent chemical shift with deprotonation of the phosphate resulting in a shift of the 31P resonance to lower fields. A single 31P NMR signal at a frequency corresponding to fully ionized phosphate monoesters is observed in aspartate-transaminase-bound pyridoxal or pyridoxamine phosphate. The 31P resonance in the holotransaminase is pH-independent and is unaffected by saturating concentrations of substrates or inhibitors. Only denaturation with 6 M guanidine with HCl results in changes in the 31P of the holoenzyme. It appears that the phosphate group of pyridoxal phosphate is bound to a positive pocket in the holoenzyme and remains fully ionized in the pH range of 5.6 to 9.2. The phosphate-binding properties are present even in the apoenzyme which is able to bind inorganic phosphate which then can be displaced by pyridoxal or pyridoxamine phosphate in the process of holoenzyme formation.
mesh_terms: D000818:Animals; D001059:Apoproteins; D001219:Aspartate Aminotransferases; D001665:Binding Sites; D003593:Cytoplasm / Q000201:enzymology; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D009206:Myocardium / Q000201:enzymology* / Q000648:ultrastructure; D009943:Organophosphorus Compounds; D010758:Phosphorus; D011485:Protein Binding; D011489:Protein Denaturation; D011730:Pyridoxal; D011733:Pyridoxamine / Q000031:analogs & derivatives*; D013552:Swine
chemical_list: D001059:Apoproteins; D006146:Guanidines; D009943:Organophosphorus Compounds; D010758:Phosphorus; D011730:Pyridoxal; D011733:Pyridoxamine; D001219:Aspartate Aminotransferases; C010627:pyridoxamine phosphate