Identification of coenzyme aldimine proton in proton NMR spectra of pyridoxal 5'-phosphate dependent enzymes: aspartate aminotransferase isoenzymes

Abstract

The pyridoxal form of the alpha subform of cytosolic aspartate aminotransferase (EC 2.6.1.1) is fully active and binds pyridoxal 5'-phosphate via an aldimine formation with Lys-258 whereas the gamma subform is virtually inactive and lacks the aldimine linkage. Comparison of 1H NMR spectra between the alpha and gamma subforms suggested that peak 1 of the alpha subform at 8.89 ppm contains a resonance assignable to the internal aldimine 4'-H. Reaction with a reagent that cleaves or modifies the internal aldimine bond [(amino-oxy)acetate, L-cysteinesulfinate, NH2OH, NaBH4, or NaCNBH3] caused the disappearance of a resonance line at 8.89 ppm that possessed a broad line width and corresponded in intensity to a single proton. These reagents were also used successfully for the identification of the aldimine 4'-H resonance in the mitochondrial isoenzyme. In contrast to the cytosolic isoenzyme whose resonance for the 4'-H did not show any detectable change in chemical shift with pH, the corresponding resonance in the mitochondrial isoenzyme exhibited pH-dependent chemical shift change (8.84 ppm at pH 5 and 8.67 ppm at pH 8) with a pK value of 6.3, reflecting the interisozymic difference in the microenvironment provided for the internal aldimine. Validity of the signal assignment was further shown by the two findings: the resonance assigned to the 4'-H emerged upon conversion of the pyridoxamine into the pyridoxal form, and the resonance appeared upon reconstitution of the apoenzyme with [4'-1H]pyridoxal phosphate but not with [4'-2H]pyridoxal phosphate.

15N Nuclear Magnetic Resonance Studies of Acid−Base Properties of Pyridoxal-5‘-Phosphate Aldimines in Aqueous Solution

“…On the other hand, the doubly negatively charged phosphate group slightly enhances the basicity of the imino nitrogen, since removal of the phosphate group in 3 leads to a decrease in the corresponding p K a from 11.4 to 10.5. The p K a values for the imino nitrogens of 2a and 3 obtained here correspond closer to the apparent p K a values of external aldimines, which are usually 9−11, − than to those of internal aldimines, which are in order of 6−7. − The electron-withdrawing effect of the imino function affects the ring nitrogen whose p K a decreases to 5.8, a normal value for pyridine derivatives. − One anticipates an even larger drop in the absence of the doubly negatively charged phosphate group. As mentioned above, in the case of PLP, the pyridine/phenolate site loses its proton only above pH 8.…”

“…The pK a values for the imino nitrogens of 2a and 3 obtained here correspond closer to the apparent pK a values of external aldimines, which are usually [9][10][11][42][43][44] than to those of internal aldimines, which are in order of 6-7. [45][46][47] The electron-withdrawing effect of the imino function affects the ring nitrogen whose pK a decreases to 5.8, a normal value for pyridine derivatives. [48][49][50] One anticipates an even larger drop in the absence of the doubly negatively charged phosphate group.…”

Porcine cytosolic aspartate aminotransferase reconstituted with [4'-13C]pyridoxal phosphate. pH and ligand-induced changes of the coenzyme observed by carbon-13 NMR spectroscopy

“…PPM figure 3: Selected region of 75.46-MHz 13C NMR spectra showing the effect of pH and 2-methyl-DL-aspartate concentration on the chemical shift and line width of C4' of the pyridoxal form of reconstituted AspAT. Protein concentration is approximately 1 mM in all samples under the following conditions of pH, Me Asp concentration, and number of scans: (A) 5.55, 0.0 mM, 65000; (B) 5.37, 10 mM, 70000; (C) 5.39, 50 mM, 75000; (D) 7.73, 0.0 mM, 290000. of the imino group (Morino et al, 1986). The line width of C4' during the course of the titration remained relatively unchanged, randomly varying from 115 to 140 Hz.…”

“…The reconstituted enzyme was maintained as the pyridoxal form (~50 mg/mL) at 4 °C in 10 mM KPB (pH 6) in the presence of 50 mM succinate to stabilize the enzyme. This procedure for preparation of the pyridoxamine form and the apoenzyme is based on that described previously (Jenkins & D'Ari, 1966a;Scardi et al, 1963;Morino et al, 1986). I3C NMR Spectroscopy.…”