Serine hydroxymethyltransferase. 31P nuclear magnetic resonance study of the enzyme-bound pyridoxal 5'-phosphate

The environment of the phosphate group of pyridoxal-P bound at the active site of cytosolic serine hydroxymethyltransferase has been investigated by 31P NMR spectroscopy. In the holoenzyme, the pyridoxal-P chemical shift is pH-dependent with a pKa of 6.45. The chemical shift of the bound pyridoxal-P is shifted upfield about 0.3 ppm from the signal for free pyridoxal-P. Saturation of the active site with the substrates L-serine, glycine, and tetrahydrofolate does not alter the chemical shift or the pKa of the phosphate group. The addition of these substrates does, however, alter the absorption and circular dichroism spectra of the bound coenzyme, reflecting environmental changes of the pyridine ring-Schiff's base system. We conclude from these studies that the phosphate group of the bound coenzyme is exposed to the solvent. The reorientation and conformational changes of the pyridoxal-P ring which take place during the formation of enzyme-substrate complexes do not appear to change the environment of the phosphate moiety of the coenzyme.