NMR spectra of exchangeable protons of pyridoxal phosphate-dependent enzymes

We have recorded 1H NMR spectra in H2O for exchangeable protons of four pyridoxal phosphate-dependent enzymes: D-serine dehydratase, aspartate aminotransferase, tryptophan: indole-lyase and glutamate decarboxylase. The molecular masses range from 48-250 kDa. In every case there are downfield peaks which are lost when the apoenzyme is formed. In most cases some peaks shift in response to interactions with substrates and inhibitors and with changes in pH. We associate one downfield resonance with the proton on the ring nitrogen of the coenzyme and others with imidazole groups that interact with coenzyme or substrates. The chemical shift for the coenzyme-bound proton differs for free enzyme, substrate Schiff base or quinonoid forms.