Studies on the interaction of ligands with phosphorylase b using a spin-label probe.

1 Phosphorylase b can be specifically spin labelled at one SH group per subunit without affecting either the specific activity or the co-operativity between the 5-AMP sites. 2 The electron spin resonance spectrum indicates that the mobility of the spin label is greater than that of the whole enzyme. This, together with measurements of the hyperfine coupling constant suggests that the spin-labelled SH is exposed to the solvent. 3 The conformational change induced by 5-AMP is detected by the spin label. This transition occurs over the same concentration range as that for activation of the enzyme, and shows similar co-operativity. 4 The previously postulated fourth conformation in the presence of 5-AMP and glucose 1-phosphate can be confirmed by a study of the spin-label spectrum in the presence of both ligands. 5 IMP and glucose have almost no effect on the spin label on the enzyme. 6 Glucose 6-phosphate causes a conformational change, and the binding of glucose 6-phosphate to the spin-labelled enzyme is hyperbolic. 7 Addition of Mn2+ to the spin-labelled enzyme results in a diminution of the electron spin resonance signal by approximately 18%, while addition of Mg2+ causes no change in the spectrum. 8 The broadening of the proton resonances of AMP, glucose 1-phosphate, glucose 6-phosphate and adenosine, resulting from interaction with the unpaired electron spin on the labelled enzyme, has been studied. 9 The distance between the unpaired electron and selected protons on these ligands have been calculated. 10 A brief outline of the calculations is given in the Appendices.