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 gamma delta)4. Previous studies have revealed that the activity of its catalytic gamma subunit is controlled by alterations in quaternary structure initiated at allosteric and covalent modification sites on PhK's three regulatory subunits; however, changes in the conformation of the holoenzyme initiated by the catalytic subunit have been more difficult to document. In this study a monoclonal antibody (mAb gamma79) has been generated against isolated gamma subunit and used as a conformational probe of that subunit. The epitope recognized by this antibody is within the catalytic core of the gamma subunit, between residues 100 and 240, and monovalent fragments of the antibody inhibit the catalytic activity of the holoenzyme, the gamma-calmodulin binary complex, and the free gamma subunit. Activation of PhK by a variety of mechanisms known or thought to act through its regulatory subunits (phosphorylation, ADP binding, or alkaline pH) increased the binding of the holoenzyme to immobilized mAb gamma79, 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 gamma79. The activating ligand Mg2+ also stimulated the binding of the PhK holoenzyme to immobilized mAb gamma79, as well as the binding of mAb gamma79 to immobilized gamma subunit. Thus, Mg2+ increases the accessibility of the mAb gamma79 epitope in both the isolated gamma subunit and in the holoenzyme. Our results suggest that previously reported influences of Mg2+ on the quaternary structure of the PhK holoenzyme are directly mediated by the gamma subunit.
Wilkinson
D A
DA
Department of Biochemistry, College of Medicine, University of Tennessee, Memphis 38163, USA.
Fitzgerald
T J
TJ
Marion
T N
TN
Carlson
G M
GM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D020134:Catalytic Domain; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005260:Female; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits
D000911:Antibodies, Monoclonal; D020035:Holoenzymes; D010764:Phosphorylase Kinase; D008274:Magnesium