The glycogen phosphorylase/ phosphorylase kinase interaction: effects of mutations in the amino-terminal region of glycogen phosphorylase

Glycogen phosphorylase is an important enzyme for carbohydrate metabolism in muscle. It uses inorganic phosphate to remove glucose from glycogen, producing glucose-1-phosphate, which can be used for the production of ATP. Inactive glycogen phosphorylase (phosphorylase h) is activated either by the allosteric binding of 5\u27-AMP, or by phosphorylation by phosphorylase kinase (PhK). Phosphorylation 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 substrate for PhK. This dissertation research has attempted to determine 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, gamma(1--300). Three mutations (R69K, R69E, and E501A), made at sites that interact with the amino terminus in either phosphorylase b or a, showed little difference in phosphorylation by gamma(1--300) compared to 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 gamma(1--300), compared to phosphorylase b. R16E was the poorest substrate for gamma(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 gamma(1--300). In addition, I13G and R16A were able to be phosphorylated by protein kinase A, which does not recognize native phosphorylase;Some of the mutants were also observed to have altered conformational states. R16A and R16E were activated at very low AMP concentration and crystallized at low temperature, like phosphorylase a. This indicates that even without phosphorylation, their structures are more like phosphorylase a than phosphorylase b. Two other mutants produced the opposite effect, behaving like phosphorylase b after phosphorylation. R69E was only partially activated by phosphorylation, and I13G was completely inactive after phosphorylation. I13G was the first observation of a phosphorylase form that could not be activated by phosphorylation