Mm. Crerar et al., CHIMERIC MUSCLE AND BRAIN GLYCOGEN PHOSPHORYLASES DEFINE PROTEIN DOMAINS GOVERNING ISOZYME-SPECIFIC RESPONSES TO ALLOSTERIC ACTIVATION, The Journal of biological chemistry, 270(23), 1995, pp. 13748-13756
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.