Fluorine-19 as a covalent active site-directed magnetic resonance probe in aspartate transaminase.
Phosphypyridoxyl trifluoroethylamine has been synthesized as an active site-directed 19F NMR probe for aspartate transaminase. This coenzyme derivative adds stoichiometrically to the apotransaminase as observed by both fluorescence and circular dichroism measurements. The fluorinated phosphypyridoxamine derivative, when bound to the apotransaminase, will not dissociate upon extensive dialysis or passage through Sephadex G-25. The compound behaves as a pyridoxamine phosphate derivative and not as a coenzyme-substrate complex, since both competing anions and dicarboxylic acid inhibitors still bind to the phosphopyridoxyl trifluoroethylamine enzyme. The 19F NMR spectra of the enzyme-bound phosphopyridoxyl trifluoroethylamine were measured as a function of pH, ionic strength, and temperature. The 19F MNR of the enzyme-bound coenzyme derivative revealed no predetermined asymmetry in the subunits of aspartate transaminase insolution in terms of differences in chemical shift or resonance line shape between the two environments. A pH-dependent chemical shift change of the single 19F resonance was observed, which is consistent with the influence of a single ionization with an apparent pKa of 8.4 in 0.10 M KCl at 30 degrees. Increasing the ionic strength resulted in increasing values for the observed pKa, the highest recorded value was 9.1 in 3.0 M KCl. The temperature dependence of the pH titration of the chemical shift gives deltaH' of ionization of 10.5 kcal/mol. The evidence suggests a possible epsilon-amino group, electrostatically affected by positive charges, being responsible for the titration effect of the active site-bound fluorine derivative of pyridoxamine phosphate.
D000818:Animals; D001051:Apoenzymes; D001219:Aspartate Aminotransferases / Q000378:metabolism; D001665:Binding Sites; D005461:Fluorine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009206:Myocardium / Q000201:enzymology; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives; D013552:Swine
D001051:Apoenzymes; D005461:Fluorine; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases