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 contains two autoinhibitory calmodulin binding domains designated PhK13 and PhK5. These peptides inhibit truncated gamma(1-300). Previous data show that PhK13 (residues 302-326) is a competitive inhibitor with respect to phosphorylase b, with a K(i) of 1.8 microm. This result suggests that PhK13 may bind to the active site of truncated gamma(1-300). Variants of PhK13 were prepared to localize the determinants for interaction with the catalytic fragment 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 microm. PhK13 has been proposed to function as a pseudosubstrate inhibitor with Cys-308 occupying the site that normally accommodates the phosphorylatable serine in phosphorylase b. A PhK13-1 variant, C308S, was synthesized. Kinetic characterization of this peptide reveals that it does not serve as a substrate but is a competitive inhibitor. Additional variants were designed based on previous knowledge of phosphorylase kinase substrate determinants. Variants were analyzed as substrates and as inhibitors for truncated gamma(1-300). Although PhK13-1 does not appear to function as a pseudosubstrate, several specificity determinants employed in the recognition of phosphorylase b as substrate are utilized in the recognition of PhK13-1 as an inhibitor.
Bartleson
C
C
Signal Transduction Training Program and the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA. cjsuitt@iastate.edu
Graves
D J
DJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D002147:Calmodulin / Q000378:metabolism; D020134:Catalytic Domain; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010766:Phosphorylation
D002147:Calmodulin; D010446:Peptide Fragments; D010764:Phosphorylase Kinase; D000251:Adenosine Triphosphatases