Abstract
Liver and muscle glycogen phosphorylases, which are products of distinct genes, are both activated by covalent phosphorylation, but in the unphosphorylated (b) state, only the muscle isozyme is efficiently activated by the allosteric activator AMP. The different responsiveness of the phosphorylase isozymes to allosteric ligands is important for the maintenance of tissue and whole body glucose homeostasis. In an attempt to understand the structural determinants of differential sensitivity of the muscle and liver isozymes to AMP, we have developed a bacterial expression system for the liver enzyme, allowing native and engineered proteins to be expressed and characterized. Engineering of the single amino acid substitutions Thr48Pro, Met197Thr and the double mutant Thr48Pro, Met197Thr in liver phosphorylase, and Pro48Thr in muscle phosphorylase, did not qualitatively change the response of the two isozymes to AMP. These sites had previously been implicated in the configuration of the AMP binding site. However, when nine amino acids among the first 48 in liver phosphorylase were replaced with the corresponding muscle phosphorylase residues (L1M2-48L49-846), the engineered liver enzyme was activated by AMP to a higher maximal activity than native liver phosphorylase. Interestingly, the homotropic cooperativity of AMP binding was unchanged in the engineered phosphorylase b protein, and heterotropic cooperativity between the glucose-1-phosphate and AMP sites was only slightly enhanced. The native liver, native muscle and L1M2-48L49-846 phosphorylases were converted to the a form by treatment with purified phosphorylase kinase; the maximal activity of the chimeric a enzyme was greater than the native liver a enzyme and approached that of muscle phosphorylase a. From these results we suggest that tissue-specific phosphorylase isozymes have evolved a complex mechanism in which the N-terminal 48 amino acids modulate intrinsic activity (Vmax), probably by affecting subunit interactions, and other, as yet undefined regions specify the allosteric interactions with ligands and substrates.
Highlights
Liver and muscleglycogen phosphorylases, which Mammalian glycogenolysis is catalyzed by a family of glyare products of distinct genes, are both activated by cogen phosphorylases that includes isozymes designated as covalent phosphorylation, but in thuenphosphorylated muscle, liver, or brain according to the tissue in which they ( b ) state, only the muscle isozyme is efficiently acti- are preferentially expressed
From these results we suggest that tissue-specific AMP binding site (Thr4’Pro) or in altering subunit contacts phosphorylase isozymeshave evolved a complex mech- (Thrlg7Met)(18)
Crude bacterial lysates containing native and engineered phosphorylases were resolved on native polyacrylamide gels and soaked in one of three buffers containing phosphorylase substrates and activators, as described under "Materials and Methods." As shown in Fig. 2, native liver and muscle phosphorylases expressed in bacteria exhibit the same pattern of response to theallosteric effector AMP as do the enzymes purified from tissue sources
Summary
Even at a distanceof 12 A from the AMP binding site, only nine of fied phosphorylase kinase; the maximal activity of the 77 amino acids differ between liver and muscle phosphorylase. Chimeric a enzyme was greater than thenative livera Of these, modeling suggested that two could be important in enzyme and approached that of muscle phosphorylase affecting the configuration of the elements that make up the a From these results we suggest that tissue-specific AMP binding site (Thr4’Pro) or in altering subunit contacts phosphorylase isozymeshave evolved a complex mech- (Thrlg7Met)(18). The transcription inducer isopropylthiogalactoside (Sigma), was added to a final
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