Mg2+ induces conformational changes in the catalytic subunit of phosphorylase kinase, whether by itself or as part of the holoenzyme complex

Phosphorylase kinase (PhK) from skeletal muscle is a structurally complex, highly regulated, hexadecameric enzyme of subunit composition (alpha beta g amma delta)(4). Previous studies have revealed that the activity of its cat alytic gamma subunit is controlled by alterations in quaternary structure i nitiated at. allosteric and covalent modification sites on PhK's three regu latory subunits; however, changes in the conformation of the holoenzyme ini tiated by the catalytic subunit have been more difficult to document. In th is study a monoclonal antibody (mAb gamma 79) has been generated against is olated gamma subunit and used as a conformational probe of that subunit. Th e epitope recognized by this antibody is within the catalytic core of the g amma subunit, between residues 100 and 240, and monovalent fragments of the antibody inhibit the catalytic activity of the holoenzyme, the gamma-calmo dulin binary complex, and the free gamma subunit. Activation of PhK by a va riety of mechanisms known or thought to act through its regulatory subunits (phosphorylation, ADP binding, or alkaline pH) increased the binding of th e holoenzyme to immobilized mAb gamma 79, indicating that activation by any of these distinct mechanisms involves repositioning of the portion of the catalytic domain of the gamma subunit containing the epitope for mAb gamma 79. The activating ligand Mg2+ also stimulated the binding of the PhK holoe nzyme to immobilized mAb gamma 79, as well as the binding of mAb gamma 79 t o immobilized gamma subunit. Thus, Mg2+ increases the accessibility of the mAb gamma 79 epitope in both the isolated gamma subunit and in the holoenzy me. Our results suggest that previously reported influences of Mg2+ on the quaternary structure of the PhK holoenzyme are directly mediated by the gam ma subunit.