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.
Nadeau
O W
OW
Department of Biochemistry, College of Medicine, University of Tennessee, Memphis 38163, USA.
Traxler
K W
KW
Fee
L R
LR
Baldwin
B A
BA
Carlson
G M
GM
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