INTERSUBUNIT TRANSMISSION OF LIGAND EFFECTS IN THE GLYCOGEN PHOSPHORYLASE-B DIMER

The heterotropic interactions between AMP, the allosteric activator, and glucose 6-phosphate, an inhibitor, binding to glycogen phosphorylase b have been studied by a novel procedure. The affinity label 8-[m-(m-fluorosulfo-nylbenzamido)benzylthio]adenine has been shown to mimic the action of bound AMP when reacted with AMP binding sites on each monomer of the phosphorylase b dimer [Anderson, R. A, Parrish, R. F., & Graves, D. J. (1973) Biochemistry 12, 1895-1900]. The affinity label activates the enzyme and prevents AMP and glucose 6-phosphate binding, as probed by a spin-label and catalytic activity. A stable hybrid dimer, having only one monomer modified with the affinity label, has been isolated by using 5'-AMP-Sepharose affinity chromatography. The activity of the hybrid in the absence of AMP was a measure of the activity of the modified subunit only, while addition of AMP activated the unmodified subunit only. Glucose 6-phosphate binds only to the unmodified monomer, yet it inhibits the activity (in the absence of AMP) of the modified subunit, showing heterotropic interactions across the subunit interface. The dissociation constant for glucose 6-phosphate binding to the unmodified monomer was an order of magnitude higher than that for binding to native spin-labeled enzyme. This demonstrated that the affinity label modification also affects the unmodified subunit of the hybrid. Such interactions probably occur in vivo between AMP and glucose 6-phosphate binding to different subunits. Glucose 6-phosphate/AMP competition within one subunit was simply investigated by the use of the +AMP activity of the unmodified subunit of the hybrid which lacks the AMP homotropic binding properties of the native enzyme. The intrasubunit interaction was shown to be partially competitive. © 1979, American Chemical Society. All rights reserved.