Site-directed mutants of glycogen phosphorylase are altered in their interaction with phosphorylase kinases

Glycogen phosphorylase is found in resting muscle as phosphorylase b, which is inactive without AMP. Phosphorylation by phosphorylase kinase (PhK) pro duces phosphorylase a, which is active in the absence of AMP. PhK is the on ly kinase that can phosphorylate phosphorylase b, which in turn is the only physiological substrate for PhK. We have explored the reasons for this spe cificity and how these two enzymes recognize each other by studying site-di rected mutants of glycogen phosphorylase. All mutants were assayed for chan ges in their interaction with a truncated form of the catalytic subunit of phosphorylase kinase, gamma>(*) over bar * (1-300). Five mutations (R69K, R 69E, R43E, R43E/R69E, and E501A), made at sites that interact with the amin o terminus in either phosphorylase b or a, showed little difference in phos phorylation by gamma>(*) over bar * (1-300) compared to wild-type phosphory lase b. Five mutations, made at three sites in the aminoterminal tail of ph osphorylase (K11A, K11E, I13G, R16A, and R16E), however, produced decreases in catalytic efficiency for gamma>(*) over bar * (1-300), compared to that for phosphorylase b. R16E was the poorest substrate for gamma>(*) over bar * (1-300), giving a 47-fold decrease in catalytic efficiency. The amino te rminus, and especially Arg 16, are very important factors for recognition o f phosphorylase by gamma>(*) over bar * (1-300). A specific interaction bet ween Lys 11 of phosphorylase and Glu 110 of gamma>(*) over bar * (1-300) wa s also confirmed. In addition, I13G and R16A were able to be phosphorylated by protein kinase A, which does not recognize native phosphorylase.