Proximal regions of the catalytic gamma and regulatory beta subunits on the interior lobe face of phosphorylase kinase are structurally coupled to each other and with enzyme activation.
Phosphorylase kinase from skeletal muscle is a hexadecameric enzyme with the subunit composition (alphabeta gammadelta)4 and a mass of 1.3 x 10(6) Da. The catalytic gamma subunit and the remaining regulatory subunits are packed as a tetrahedral structure composed of two elongated, opposing (alphabeta gammadelta)2 octameric lobes. We show by immunoelectron microscopy with subunit-specific monoclonal antibodies that a portion of the beta subunit occurs on the interior face of the lobes at a region of inter-lobal interactions, and that at a proximal position slightly more central and distal on the interior lobe face lies the base (residues 277 to 290) of the helical domain of the catalytic core of the gamma subunit. Activation of the kinase by a variety of means caused similar increases in the binding to the holoenzyme of the monoclonal antibodies against these two regions of the beta and gamma subunits. Moreover, monovalent fragments of the antibodies against both regions stimulated the activity of the non-activated holoenzyme. Thus, the epitopes of the beta and gamma subunits recognized by the monoclonal antibodies are structurally coupled to each other and with the activation of phosphorylase kinase. Activation of the holoenzyme apparently involves the repositioning of the base of the catalytic domain of the gamma subunit and a proximal region of the beta subunit within the identified area on the interior face of the lobes of the tetrahedral phosphorylase kinase molecule.
Wilkinson
D A
DA
Department of Biochemistry, University of Tennessee, Memphis 38163, USA.
Norcum
M T
MT
Fizgerald
T J
TJ
Marion
T N
TN
Tillman
D M
DM
Carlson
G M
GM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D005260:Female; D016253:Microscopy, Immunoelectron / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010764:Phosphorylase Kinase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits
D000911:Antibodies, Monoclonal; D003067:Coenzymes; D000939:Epitopes; D010764:Phosphorylase Kinase