Activators of phosphorylase kinase alter the cross-linking of its catalytic subunit to the C-terminal one-sixth of its regulatory alpha subunit.
Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer consisting of four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha, beta, and delta, the last being endogenous calmodulin). The enzyme is activated by a variety of effectors acting through its regulatory subunits. To probe the quaternary structure of nonactivated and activated forms of the kinase, we used the heterobifunctional, photoreactive cross-linker N-5-azido-2-nitrobenzoyloxysuccinimide. Mono-derivatization of the holoenzyme with the succinimidyl group, followed by photoactivation of the covalently attached azido group, resulted in intramolecular cross-linking to form two distinct heterodimers: a major (alphagamma) and a minor (betadelta) conjugate. Formation of both conjugates was significantly altered in activated conformations of the enzyme induced by phosphorylation, alkaline pH, and several allosteric activators (ADP, exogenous calmodulin/Ca2+, and Ca2+ alone). Of these activating mechanisms, all increased formation of alphagamma, except Ca2+ alone, which inhibited its formation. When cross-linking was carried out at alkaline pH or in the presence of ADP or exogenous calmodulin/Ca2+, the cross-linked enzyme remained activated following removal of the activators; however, cross-linking in the presence of Ca2+ resulted in sustained inhibition. The results indicate that perturbations in the subunit cross-linking forming the alphagamma dimer reflect the subsequent extent of sustained activation of the holoenzyme that is measured. The region cross-linked to the catalytic gamma subunit was confined to the C-terminal 1/6th of the alpha subunit, which contains known regulatory regions. These results suggest that activators of the phosphorylase kinase holoenzyme perturb interactions between the C-terminal region of the inhibitory alpha subunit and the catalytic gamma subunit, ultimately leading to activation of the latter.
Department of Biochemistry, College of Medicine, University of Tennessee, Memphis 38163, USA.
O W
OW
Nadeau
K W
KW
Traxler
L R
LR
Fee
B A
BA
Baldwin
G M
GM
Carlson
D000818:Animals; D001386:Azides / Q000737:chemistry; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D018656:Muscle Fibers, Fast-Twitch / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D010764:Phosphorylase Kinase / Q000737:chemistry* / Q000378:metabolism*; D010777:Photochemistry; D011487:Protein Conformation; D011817:Rabbits; D013388:Succinimides / Q000737:chemistry
D001386:Azides; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D013388:Succinimides; C049915:N-hydroxysuccinimidyl-5-azido-2-nitrobenzoate; D010764:Phosphorylase Kinase