Abstract
Using site-directed mutagenesis, we proposed that an autoinhibitory domain(s) is located at the C-terminal region (301-386) of the phosphorylase 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). Removal of the putative inhibitory domain(s) by truncation results in the generation of a constitutively active and calmodulin-independent form, gamma 1-300. To probe the structural basis of autoinhibition of gamma-subunit activity, two synthetic peptides, PhK13 (gamma 303-327) and PhK5 (gamma 343-367), corresponding to the two calmodulin-binding regions, were assayed for their ability to inhibit gamma 1-300. Competitive inhibition of gamma 1-300 by PhK13 was found versus phosphorylase b (Ki = 1.8 microM) 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 auto-inhibitory domains within the C terminus of the gamma-subunit and that these two domains overlap with the calmodulin-binding regions. Two mutant forms of gamma 1-300, E111K and E154R, were used to probe the enzyme-substrate-binding region using peptide substrate analogs corresponding to residues 9-18 of phosphorylase b (KRK11Q12ISVRGL). The data suggest that Glu111 interacts with the P-3 position of the substrate (Lys11) and Glu154 interacts with the P-2 site (Gln12). Both E111K and E154R were competitively inhibited with respect to phosphorylase b by PhK13, with 14- and 8-fold higher Ki values, respectively, than that observed with the wild-type enzyme. These data are consistent with a model for the regulation of the gamma-subunit of phosphorylase kinase in which PhK13 acts as a competitive pseudosubstrate that directly binds the substrate binding site of the gamma-subunit (Glu111 and Glu154).
Highlights
EXPERIMENTAL PROCEDURESMaterials-[ y_32P]ATP was purchased from ICN Biomedicals. Other reagents were purchased from Sigma
From the Wepartment of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011 and the IlDepartment of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah 84112
Kinetic Analyses of the Inhibition of 1'1--300 by PhK13 and PhK5-Kinetic analyses were undertaken to determine the mechanism of the inhibitory properties of PhK13 and PhK5 on 1'1-300' The results of these analyses showed that PhK13 is a competitive inhibitor of phosphorylase b phosphorylation and a noncompetitive inhibitor of ATP (Table I and Fig. 3)
Summary
Materials-[ y_32P]ATP was purchased from ICN Biomedicals. Other reagents were purchased from Sigma. Activity Assay-The protein kinase activity of the y-subunit was determined by incorporation of 3Zp into phosphorylase b as described previously with some modification [5, 22]. The standard assay was performed at 30 DC for 5 min and contained final concentrations of 60. 1 and 2), the standard assay was used except PhK13 or PhK5 were added and the final concentrations of phosphorylase band Yl-300 were adjusted to 4 fJoM and 1 fJoglml, respectively. The standard assay mixture as described above containing PhK13 (10 fJoM) or PhK5 (250 fJoM) was first incubated at 30 DC for 5 min. The inhibition data (Table I) with PhK13 and PhK5 were fitted to the equations for competitive or noncompetitive inhibition with the program Enzfitter. All the kinetic analyses were repeated at least three times
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