Open AccessStudies on the Interaction of Ligands with Phosphorylase b Using a Spin‐Label Probe
Author(s) -
Campbell Iain D.,
Dwek Raymond A.,
Price Nicholas C.,
Radda George K.
Publication year - 1972
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1972.tb02103.x
Subject(s) - electron paramagnetic resonance , chemistry , spin label , enzyme , spin (aerodynamics) , unpaired electron , conformational change , resonance (particle physics) , crystallography , hyperfine structure , nuclear magnetic resonance , stereochemistry , biochemistry , atomic physics , physics , engineering , aerospace engineering
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 Mn 2+ to the spin‐labelled enzyme results in a diminution of the electron spin resonance signal by approximately 18%, while addition of Mg 2+ 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.