Muscle glycogen phosphorylase a contains a small proportion of its coenzyme, pyridoxal phosphate, bound in a Schiff base form which absorbs at 415 nm. Excited at this wavelength, it fluoresces with two maxima, 510 nm and 535–540 nm. The ratio of the latter peak height to that of 510 nm varies directly with the specific catalytic activity. Experiments on phosphorylase a with increasing concentrations of imidazole citrate show very similar results. When interpreted in analogy to solvent perturbation effects on model compounds, this finding suggests that the catalytic efficiency depends on the degree of hydrophobicity of the pyridoxal phosphate environment, and provides one more link between the coenzyme and the catalytic process.A single large crystal of phosphorylase a, when excited at 330 nm, exhibits nearly half of its fluorescence emission at 415 nm and the remainder at 510–535 nm, whereas in solution only a small amount of fluorescence is seen at 415 nm. Furthermore, decreasing the temperature of the crystal increases the proportion of fluorescence emission at 415 nm. These results are interpreted on the basis of the hypothesis that the coenzyme is bound in the form of a carbinol amine, absorbing at 330 nm, which is split on excitation to form a Schiff base which emits as such at 535 nm. The crystal structure of the protein requires more energy to effect the transition to the Schiff base, so that a large proportion of the coenzyme molecules fluoresce as the carbinol amine form, while decreasing the temperature increases this proportion even further.
A physical-chemical study of pyridoxal phosphate in glycogen phosphorylase
