Identification of the Substrate and Pseudosubstrate Binding Sites of Phosphorylase Kinase -Subunit

• Published in: The Journal of biological chemistry Vol. 270; no. 13; p. 7183
• Main Authors: Chi-Ying F. Huang, Chiun-Jye Yuan, Donald K. Blumenthal, Donald J. Graves
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 31.03.1995
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.13.7183

Using site-directed mutagenesis, we proposed that an autoinhibitory domain(s) is located at the C-terminal region (301-386) of the phosphorylase kinase -subunit (Huang, C.-Y. F., Yuan, C.-J., Livanova, N. B., and Graves, D. J. (1993) Mol. Cell. Biochem. 127/128, 7-18). Removal of the putative inhibitory domain(s) by truncation results in the generation of a constitutively active and calmodulin-independent form, . To probe the structural basis of autoinhibition of -subunit activity, two synthetic peptides, PhK13 ( ) and PhK5 ( ), corresponding to the two calmodulin-binding regions, were assayed for their ability to inhibit . Competitive inhibition of by PhK13 was found versus phosphorylase b ( K = 1.8 μM) and noncompetitive inhibition versus ATP. PhK5 showed 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 within the C terminus of the -subunit and that these two domains overlap with the calmodulin-binding regions. Two mutant forms of , E111K and E154R, were used to probe the enzyme-substrate-binding region using peptide substrate analogs corresponding to residues 9-18 of phosphorylase b (KRK Q ISVRGL). The data suggest that Glu interacts with the P-3 position of the substrate (Lys ) and Glu interacts with the P-2 site (Gln ). Both E111K and E154R were competitively inhibited with respect to phosphorylase b by PhK13, with 14- and 8-fold higher K values, respectively, than that observed with the wild-type enzyme. These data are consistent with a model for the regulation of the -subunit of phosphorylase kinase in which PhK13 acts as a competitive pseudosubstrate that directly binds the substrate binding site of the -subunit (Glu and Glu ).