Characterization of the regulatory domain of the γ-subunit of phosphorylase kinase

Abstract

Phosphorylase kinase is a multimeric protein kinase (α₄β₄γ₄δ₄) whose enzymatic activity is conferred by its γ-subunit. A library of 18 overlapping synthetic peptides spanning residues 277-386 of the γ-subunit has been prepared to use in identifying important regulatory structures in the protein. In the present study, the library was screened to identify regions that might function as autoinhibitory domains. Peptides from two distinct regions were found to inhibit the Ca²⁺-activated holoenzyme. The same regions were previously found to bind calmodulin (i.e. the δ-subunit; Dasgupta, M. Honeycutt, T. and Blumenthal, D. K.(1989) J. Biol. Chem. 264, 17156-17163). The most potent substrate antagonist peptides were PhK13 (residues 302-326; K_i = 300 nM) and PhK5 (residues 342-366; K_i = 20 μM). Both peptides inhibited the holoenzyme competitively with respect to phosphorylase b and noncompetitively with respect to Mg•ATP. When the pattern of inhibition with both peptides present was analyzed, inhibition was observed to be synergistic and modestly cooperative indicating that the two peptides can simultaneously occupy the protein substrate-binding site(s). These data are consistent with a model in which the regions of the γ-subunit represented by PhK5 and PhK13 work in concert as regulatory subdomains that transduce Ca²⁺-induced conformational changes in the δ-subunit to the catalytic γ-subunit through a pseudosubstrate autoinhibitory mechanism.