Site-Directed Mutants of Glycogen Phosphorylase Are Altered in Their Interaction with Phosphorylase Kinase

• Published in: Biochemistry (Easton) 2000-12, Vol.39 (51), p.15887-15894
• Main Authors: Biorn, Alyssa C, Bartleson, Cheryl, Graves, Donald J
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution - genetics; Animals; Arginine - genetics; Arginine - metabolism; Cyclic AMP-Dependent Protein Kinases - metabolism; Glutamic Acid - genetics; Glutamic Acid - metabolism; Kinetics; Lysine - genetics; Muscle, Skeletal - enzymology; Mutagenesis, Site-Directed; Peptide Fragments - genetics; Peptide Fragments - metabolism; Phosphorylase b - metabolism; Phosphorylase Kinase - metabolism; Phosphorylases - genetics; Phosphorylases - metabolism; Phosphorylation; Rabbits; Recombinant Proteins - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi001755l

Glycogen phosphorylase is found in resting muscle as phosphorylase b, which is inactive without AMP. Phosphorylation by phosphorylase kinase (PhK) produces phosphorylase a, which is active in the absence of AMP. PhK is the only kinase that can phosphorylate phosphorylase b, which in turn is the only physiological substrate for PhK. We have explored the reasons for this specificity and how these two enzymes recognize each other by studying site-directed mutants of glycogen phosphorylase. All mutants were assayed for changes in their interaction with a truncated form of the catalytic subunit of phosphorylase kinase, γ(1−300). Five mutations (R69K, R69E, R43E, R43E/R69E, and E501A), made at sites that interact with the amino terminus in either phosphorylase b or a, showed little difference in phosphorylation by γ(1−300) compared to wild-type phosphorylase b. Five mutations, made at three sites in the amino-terminal tail of phosphorylase (K11A, K11E, I13G, R16A, and R16E), however, produced decreases in catalytic efficiency for γ(1−300), compared to that for phosphorylase b. R16E was the poorest substrate for γ(1−300), giving a 47-fold decrease in catalytic efficiency. The amino terminus, and especially Arg 16, are very important factors for recognition of phosphorylase by γ(1−300). A specific interaction between Lys 11 of phosphorylase and Glu 110 of γ(1−300) was also confirmed. In addition, I13G and R16A were able to be phosphorylated by protein kinase A, which does not recognize native phosphorylase.