Fluorescence of Glycogen Phosphorylase b

Abstract

The fluorescence properties of glycogen phosphorylase b and its apoenzyme are described. At neutral pH the enzyme has two fluorescence bands: one caused by the protein moiety (maximum at 335 nm, quantum yield 0.12) and the other associated with the cofactor, pyridoxal 5'-phosphate (maximum at 535 nm, quantum yield 0.012). Pyridoxal 5'-phosphate can be regarded as a native reporter group of phosphorylase. Therefore changes in its fluorescence as a function of pH made it possible to detect structural changes at the cofactor site which occurred around pH 5.3 and are not reflected in the fluorescence of the protein moiety or in the absorbance of the cofactor itself. On the other hand, the fluorescence of the protein moiety has a constant intensity in the pH range of 5 to 9.5. This plateau region of the curve coincides with the stability zone of the enzyme. With the protein fluorescence of the enzyme and the fluorescence of its cofactor, it was also possible to show the occurrence of transfer of excitation energy from the protein moiety to the cofactor (efficiency: 33%), the accessibility of water (or protons) to the pyridoxal 5'-phosphate site and to study structural relationships between phosphorylase and its apoenzyme.