title: 13C NMR spectroscopy of labeled pyridoxal 5'-phosphate. Model studies, D-serine dehydratase, and L-glutamate decarboxylase.
abstract: Pyridoxal 5'-phosphate labeled to the extent of 90% with 13C in the 4' (aldehyde) and 5' (methylene) positions has been synthesized. 13C NMR spectra of this material and of natural abundance pyridoxal 5'-phosphate are reported, as well as 13C NMR spectra of the Schiff base formed by reaction of pyridoxal 5'-phosphate with n-butylamine, the secondary amine formed by reduction of this Schiff base, the thiazolidine formed by reaction of pyridoxal 5'-phosphate with cysteine, the hexahydropyrimidine formed by reaction of pyridoxal 5'-phosphate with 1,3-diaminobutane, and pyridoxamine 5'-phosphate. The range of chemical shifts for carbon 4' in these compounds is more than 100 ppm, and thus this chemical shift is expected to be a sensitive indicator of structure in enzyme-bound pyridoxal 5'-phosphate. The chemical shift of carbon 5', on the other hand, is insensitive to these structure changes. 13C NMR spectra have been obtained at pH 7.8 and 9.4 for D-serine dehydratase (Mr = 46,000) containing natural abundance pyridoxal 5'-phosphate and containing 13C-enriched pyridoxal 5'-phosphate. The enriched material contains two new resonances not present in the natural abundance material, one at 167.7 ppm with a linewidth of approximately 24 Hz, attributed to carbon 4' of the Schiff base in the bound coenzyme, and one at 62.7 Hz with a linewidth of approximately 48 Hz attributed to carbon 5' of the bound Schiff base. A large number of resonances due to individual amino acids are assigned. The NMR spectrum changes only slightly when the pH is raised to 9.4. The widths of the two enriched coenzyme resonances indicate that the coenzyme is rather rigidly bound to the enzyme but probably has limited motional freedom relative to the protein. 13C NMR spectra have been obtained for L-glutamate decarboxylase containing natural abundance pyridoxal 5'-phosphate and 13C-enriched pyridoxal 5'-phosphate. Under conditions where the two enriched 13C resonances are clearly visible in D-serine dehydratase, no resonances are visible in enriched L-glutamate decarboxylase, presumably because the coenzyme is rigidly bound to the protein and the 300,000 molecular weight of this enzyme produces very short relaxation times for the bound coenzyme and thus very broad lines.
mesh_terms: D001665:Binding Sites; D002262:Carboxy-Lyases; D005968:Glutamate Decarboxylase; D012695:L-Serine Dehydratase; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D011732:Pyridoxal Phosphate
chemical_list: D011732:Pyridoxal Phosphate; D002262:Carboxy-Lyases; D005968:Glutamate Decarboxylase; D012695:L-Serine Dehydratase