IDENTIFICATION OF THE SUBSTRATE AND PSEUDOSUBSTRATE BINDING-SITES OF PHOSPHORYLASE-KINASE GAMMA-SUBUNIT

Using site directed mutagenesis, we proposed that an autoinhibitory do main(s) is located at the C-terminal region (301-388) of the phosphory lase kinase gamma-subunit (Huang, C.-Y.F., Yuan, C.-J., Livanova, N. B ., and Graves, D. J. (1993) Mol. Cell. Biochem. 127/128, 7-18). Remova l of the putative inhibitory domain(s) by truncation results in the ge neration of a constitutively active and calmodulin-independent form, g amma(1-300). To probe the structural basis of autoinhibition of gamma- subunit activity, two synthetic peptides, PhK13 (gamma(303-327)) and P hK5 (gamma(343-367)), corresponding to the two calmodulin-binding regi ons, were assayed for their ability to inhibit gamma(1-300). Competiti ve inhibition of gamma(1-300) by PhK13 was found versus phosphorylase b (K-i = 1.8 mu M) and noncompetitive inhibition versus ATP, PhK5 show ed noncompetitive inhibition with respect to both phosphorylase b and ATP. Calmodulin released the inhibition caused by both peptides. These results indicate that there are two distinct autoinhibitory domains w ithin the C terminus of the gamma-subunit and that these two domains o verlap with the calmodulin-binding regions. Two mutant forms of gamma( 1-300), E111K and E154R, were used to probe the enzyme-substrate-bindi ng region using peptide substrate analogs corresponding to residues 9- 18 of phosphorylase b (KRK(11)Q(12)ISVRGL). The data suggest that Glu( 111) interacts with the P-3 position of the substrate (Lys(11)) and Gl u(154) interacts with the P-2 site (Gln(12)). Both E111K and E154R wer e competitively inhibited with respect to phosphorylase b by PhK13, wi th 14- and 8-fold higher K-i values, respectively, than that observed with the wildtype enzyme. These data are consistent with a model for t he regulation of the gamma-subunit of phosphorylase kinase in which Ph K13 acts as a competitive pseudosubstrate that directly binds the subs trate binding site of the gamma-subunit (Glu(111) and Glu(154)).