Chimeric muscle and brain glycogen phosphorylases define protein domains governing isozyme-specific responses to allosteric activation.

Muscle and brain glycogen phosphorylases differ in their responses to activation by phosphorylation and AMP. The muscle isozyme is potently activated by either phosphorylation or AMP. In contrast, the brain isozyme is poorly activated by phosphorylation and its phosphorylated a form is more sensitive to AMP activation when enzyme activity is measured in substrate concentrations and temperatures encountered in the brain. The nonphosphorylated b form of the brain isozyme also differs from the muscle isozyme b form in its stronger affinity and lack of cooperativity for AMP. To identify the structural determinants involved, six enzyme forms, including four chimeric enzymes containing exchanges in amino acid residues 1-88, 89-499, and 500-842 (C terminus), were constructed from rabbit muscle and human brain phosphorylase cDNAs, expressed in Escherichia coli, and purified. Kinetic analysis of the b forms indicated that the brain isozyme amino acid 1-88 and 89-499 regions each contribute in an additive fashion to the formation of an AMP site with higher intrinsic affinity but weakened cooperativity, while the same regions of the muscle isozyme each contribute to greater allosteric coupling but weaker AMP affinity. Kinetic analysis of the a forms indicated that the amino acid 89-499 region correlated with the reduced response of the brain isozyme to activation by phosphorylation and the resultant increased sensitivity of the a form to activation by saturating levels of AMP. This isozyme-specific response also correlated with the glycogen affinity of the a forms. Enzymes containing the brain isozyme amino acid 89-499 region exhibited markedly reduced glycogen affinities in the absence of AMP compared to enzymes containing the corresponding muscle isozyme region. Additionally, AMP led to greater increases in glycogen affinity of the former set of enzymes. In contrast, phosphate affinities of all a forms were similar in the absence of AMP and increased approximately the same extent in AMP. The potential importance of a number of isozyme-specific substitutions in these sequence regions is discussed.