Correlation between activity and dynamics of the protein matrix of phosphorylase b.
Quenching of the tryptophan fluorescence of phosphorylase b was studied by using iodide and acrylamide. Steady-state measurements indicated that all indole side chains were accessible to the nonionic quencher, although only 3 out of the total of 12 residues could be quenched by I-. From Stern--Volmer plots and the fluorescence lifetime data, it was concluded that the quenching was mainly of dynamic character. The value of the collisional quenching rate constant was found to be an order of magnitude less than that obtained in the case of fully exposed tryptophans. The relatively high activation energy, 30.9 kJ/mol, of the diffusion-controlled process and the value of the activation entropy suggest that the diffusion takes place in a fluctuating, structured medium. In spite of the application of sensitive fluorescent techniques, no gross conformational changes were found in the presence of acrylamide. However, the catalytic rate of the glycogen synthesis was decreased with the residual activity of the enzyme, proportional to the concentration of the probe. Binding of activator (AMP) and substrates (glucose 1-phosphate and glycogen) was found to be unaffected by acrylamide in concentrations applied (0--0.8 M). In a similar manner, activation enthalpy did not change in the presence of the quencher either. The complete reversibility of both activity inhibition and fluorescence quenching ruled out the irreversible denaturation of the enzyme or the covalent modification of any of the functional groups. We concluded that a model, suggesting the cross-correlation of activity and fluctuation, was consistent with the experimental findings.
D000178:Acrylamides / Q000494:pharmacology; D000818:Animals; D007454:Iodides / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D009132:Muscles / Q000201:enzymology; D010763:Phosphorylase b / Q000378:metabolism*; D006005:Phosphorylases / Q000378:metabolism*; D011489:Protein Denaturation; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000178:Acrylamides; D007454:Iodides; D008024:Ligands; D010763:Phosphorylase b; D006005:Phosphorylases