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 iso zyme is poorly activated by phosphorylation and its phosphorylated a f orm is more sensitive to AMP activation when enzyme activity is measur ed in substrate concentrations and temperatures encountered in the bra in. The nonphosphorylated b form of the brain isozyme also differs fro m the muscle isozyme b form in its stronger affinity and lack: of coop erativity for AMP. To identify the structural determinants involved, s ix enzyme forms, including four chimeric enzymes containing exchanges in amino acid residues 1-88, 89-499, and 500-842 (C terminus), were co nstructed from rabbit muscle and human brain phosphorylase cDNAs, expr essed in Escherichia coli, and purified. Kinetic analysis of the b for ms 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 sit e with higher intrinsic affinity but weakened cooperativity, while the same regions of the muscle isozyme each contribute to greater alloste ric coupling but weaker AMP affinity. Kinetic analysis of the a forms indicated that the amino acid 89-499 region correlated with the reduce d response of the brain isozyme to activation by phosphorylation and t he resultant increased sensitivity of the a form to activation by satu rating levels of AMP. This isozyme specific response also correlated w ith 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 co rresponding muscle isozyme region. Additionally, AMP led to greater in creases in glycogen affinity of the former set of enzymes. In contrast , phosphate affinities of all a forms were similar in the absence of A MP and increased approximately the same extent in AMP. The potential i mportance of a number of isozyme-specific substitutions in these seque nce regions is discussed.