An inhibitory segment of the catalytic subunit of phosphorylase kinase does not act as a pseudosubstrate

The C terminus of the catalytic gamma subunit of phosphorylase kinase conta ins two autoinhibitory calmodulin binding domains designated PhK13 and PhK5 . These peptides inhibit truncated gamma (1-300). Previous data show that P hK13 (residues 302-326) is a competitive inhibitor with respect to phosphor ylase b, with a K-i of 1.8 muM (1). This result suggests that PhK13 may bin d to the active site of truncated gamma (1-300). Variants of PhK13 were pre pared to localize the determinants for interaction with the catalytic fragm ent gamma (1-300). PhK13-1, containing residues 302-312, was found to be a competitive inhibitor with respect to phosphorylase b with a K-i of 6.0 muM . PhK13 has been proposed to function as a pseudosubstrate inhibitor with C ys-308 occupying the site that normally accommodates the phosphorylatable s erine in phosphorylase b. A PhK13-1 variant, C308S, was synthesized. Kineti c characterization of this peptide reveals that it does not serve as a subs trate but is a competitive inhibitor. Additional variants were designed bas ed on previous knowledge of phosphorylase kinase substrate determinants. Va riants were analyzed as substrates and as inhibitors for truncated gamma (1 -300). Although PhK13-1 does not appear to function as a pseudosubstrate, s everal specificity determinants employed in the recognition of phosphorylas e b as substrate are utilized in the recognition of PhK13-1 as an inhibitor .