Conformational Changes in Glycogen Phosphorylase Studied with a Spin-Label Probe

Abstract

Phosphorylase b and a [rabbit sheletal muscle; EC 2.4.1.1] were covalently modified on essentially one -SH group per subunit by a spin label, 4-(2-iodacetamido)2,2,6,6-tetramethyl piperidinyloxyl. The labelled enzyme is fully active and exhibits all the characteristics of the native molecule. The ESR spectrum of the label depends on the nature of the ligand that is bound to the enzyme. This property of the spin label is used to study the interaction between the enzyme (both in the b and a forms) and activators (AMP, IMP, CMP), inhibitors (ADP, ATP, UDPG, glucose 6-phosphate), substrates (phosphate and glucose 1-phosphate) and other ligands (adenosine, .beta.-glycerol-2-phosphate). The interactions are analyzed in terms of the apparent ligand dissociation constants and the multiplicity of conformations that this regulatory enzyme exhibits.