Dynamic interaction between functional groups in the active site of glycogen phosphorylase b.
The quenching of coenzyme fluorescence in glycogen phosphorylase b is reinvestigated. Data with anionic quenchers show deviations from the original Stern-Volmer kinetics. A kinetic analysis based on measured lifetime data indicates a collisional quenching process, which is, however, not diffusion-controlled. It is proposed, that the quenching takes place primarily by enzyme-bound quencher species. The observed inhibition of the enzyme reaction by I- and IO-3 is consistent with this hypothesis. The inhibition pattern and spectral investigation refer to a true competition with the substrate, glucose-1-phosphate. So, this dynamic quenching can be regarded as an indicator of rapid conformational fluctuations which bring the two important active-site groups in contact. Effect of ligand binding on the quenching of coenzyme fluorescence should also be revaluated according to these results.
D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D008027:Light; D009132:Muscles / Q000201:enzymology*; D010763:Phosphorylase b / Q000378:metabolism*; D006005:Phosphorylases / Q000378:metabolism*; D011817:Rabbits; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D010763:Phosphorylase b; D006005:Phosphorylases