PROXIMAL REGIONS OF THE CATALYTIC GAMMA-SUBUNITS 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 wi th the subunit composition (alpha beta gamma delta)(4) and a mass of 1 .3 x 10(6) Da. The catalytic gamma subunit and the remaining regulator y subunits are packed as a tetrahedral structure composed of two elong ated, opposing (alpha beta gamma delta)(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 l ies the base (residues 277 to 290) of the helical domain of the cataly tic core of the gamma subunit. Activation of the kinase by a variety o f means caused similar increases in the binding to the holoenzyme of t he monoclonal antibodies against these two regions of the beta and gam ma 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 mo noclonal antibodies are structurally coupled to each other and with th e activation of phosphorylase kinase. Activation of the holoenzyme app arently 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 tetrahed ral phosphorylase kinase molecule. (C) 1997 Academic Press Limited