A model for activation of the hexadecameric phosphorylase kinase complex deduced from zero-length oxidative crosslinking.

Abstract

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)(4) enzyme complex that upon activation by phosphorylation stimulates glycogenolysis. Due to its large size (1.3 MDa), elucidating the structural changes associated with the activation of PhK has been challenging, although phosphoactivation has been linked with an increased tendency of the enzyme's regulatory β-subunits to self-associate. Here we report the effect of a peptide mimetic of the phosphoryltable N-termini of β on the selective, zero-length, oxidative crosslinking of these regulatory subunits to form β-β dimers in the nonactivated PhK complex. This peptide stimulated β-β dimer formation when not phosphorylated, but was considerably less effective in its phosphorylated form. Because this peptide mimetic of β competes with its counterpart region in the nonactivated enzyme complex in binding to the catalytic γ-subunit, we were able to formulate a structural model for the phosphoactivation of PhK. In this model, the nonactivated state of PhK is maintained by the interaction between the nonphosphorylated N-termini of β and the regulatory C-terminal domains of the γ-subunits; phosphorylation of β weakens this interaction, leading to activation of the γ-subunits.