Isolation of an autoinhibitory region from the regulatory beta-subunit of phosphorylase kinase.

An equimolar mixture of the regulatory alpha- and beta-subunits of phosphorylase kinase has been shown to inhibit its catalytic gamma-subunit (Paudel, H.K., and Carlson, G.M. (1987) J. Biol. Chem. 262, 11912-11915). The possible presence of an autoinhibitory sequence within those regulatory subunits has been evaluated by peptide isolation and characterization following chemical and proteolytic cleavage of an isolated equimolar mixture of those subunits; the peptides generated were tested for their ability to inhibit the activity of a complex of the gamma-subunit and calmodulin. An isolated inhibitory fragment, hereafter referred to as I-peptide, was sequenced and found to correspond to residues 420-436 of the beta-subunit (KRNPGSQKRFPSNCGRD). This sequence showed homology with the kinase's natural substrate, phosphorylase b. A synthetic peptide based on this sequence was constructed and used to study the mechanism of inhibition. Kinetic analysis of the inhibition of the gamma-subunit-calmodulin complex by the I-peptide revealed a competitive pattern versus the homologous substrate phosphorylase b and an uncompetitive pattern versus MgATP, suggesting an ordered binding of substrates, with the nucleotide binding first. In addition to its ability to inhibit, the I-peptide was also a substrate for the gamma-subunit-calmodulin complex, with a relatively good Km but poor Vmax. The parallel inhibition of free gamma-subunit and the gamma-subunit-calmodulin complex by progressively increasing concentrations of I-peptide provided further evidence that the peptide inhibits by interacting directly with the catalytic subunit and not with the stimulatory calmodulin molecule. The results of this study are consistent with previous findings from this laboratory showing that the conformation of the beta-subunit changes following activation of phosphorylase kinase through a variety of mechanisms.