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

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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 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 microenvironmental provided for the internal aldimine. Validity of the signal assignment was further shown by the two findings: (a) the resonance assigned to the 4''-H emerged upon conversion of the pyridoxamine into the pyridoxal form, and (b) the resonance appeared upon reconstitution of the apoenzyme with [4''-1H] pyridoxal phosphate but not with [4''-2H] pyridoxal phosphate.