This study shows that conformational changes in glycogen phosphorylase are accompanied by changes in the accessibility of tryptophan residues and of the coenzyme, pyridoxal phosphate, to the surrounding aqueous medium. The accessibility was estimated by determining the extent to which iodide can quench the fluorescence emission of these moieties by colliding with them, since iodide cannot collide with a buried chromophore and hence cannot quench its fluorescence. Rabbit muscle phosphorylase b, its apoform, and phosphorylase a exhibit differences in the number of exposed tryptophans, while the phosphorylase b forms from rabbit skeletal muscle and pig heart also show differences.Differences are also observed in the accessibility of the coenzyme in different forms of the enzyme. The quenching rate constant, a measure of accessibility, differs for phosphorylases a and b, and this constant is affected differently by ligand binding to the two forms. While the allosteric inhibitors, ATP and glucose 6-phosphate, render the pyridoxal phosphate moiety of phosphorylase b more accessible, the activator, AMP, and substrate, glucose 1-phosphate, together cause it to be totally inaccessible to fluorescence quenching by iodide. AMP and glucose 1-phosphate appear to mediate a conformational change which buries the coenzyme. While pyridoxal phosphate is necessary for catalytic activity, one may conclude from these experiments that its ring structure is unlikely to participate directly in the catalytic mechanism.
Evidence for direct phosphate-phosphate interaction between pyridoxal phosphate and substrate in the glycogen phosphorylase catalytic mechanism.