Comparison of the absorbance spectra and fluorescence behavior of phosphorylase b with that of model pyridoxal phosphate derivatives in various solvents.

The unusual fluorescence behavior of the pyridoxal-P coenzyme of phosphorylase b, which shows on excitation at 330 nm a maximal fluorescence emission at 535 nm, could be mimicked by model compounds of pyridoxal-P and various amines in hydrophobic solvents. Study of the model compounds indicated that the pyridoxal-P and the amine initially form a carbinol amine which, upon activation, yields a Schiff base. We conclude from these experiments that phosphorylase contains a similar carbinol amine structure in which the pyridoxal-P is bound, in addition to the c-amino group of a lysine, to a second group with nucleophilic character. This carbinol amine is split by light to form a Schiff base in the excited state which re-emits at its normal maximum of 535 nm. After falling from the excited state to the ground state, the original binding of the coenzyme may be restored. Difference spectra of phosphorylase b, upon addition of substrates, activators, and inhibitors, have been compared with difference spectra of pyridoxamine phosphate in dioxane and dioxane-water mixtures, and suggest that changes in the environment of the cofactor can affect the pK of the phenolic hydroxyl group and cause a broadening of the absorption spectrum.