Identification of coenzyme aldimine proton in 1H NMR spectra of pyridoxal 5'-phosphate dependent enzymes: aspartate aminotransferase isoenzymes.
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.
Y
Y
Morino
F
F
Nagashima
S
S
Tanase
M
M
Yamasaki
T
T
Higaki
D000625:Aminooxyacetic Acid / Q000494:pharmacology; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008929:Mitochondria, Heart / Q000201:enzymology; D018377:Neurotransmitter Agents; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D013552:Swine
D007527:Isoenzymes; D018377:Neurotransmitter Agents; D000625:Aminooxyacetic Acid; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases; D003545:Cysteine; C013461:cysteine sulfinic acid