Abstract
The mechanism of yeast glycogen phosphorylase activation by covalent phosphorylation involves structural elements distinct from the mammalian homologs. To understand the role of the amino-terminal 39-residue extension in the phosphorylation control mechanism, mutants with 22 and 42 amino-terminal residues removed were expressed in Escherichia coli, and their properties were compared with the wild-type (WT) enzyme. The unphosphorylated WT enzyme had a specific activity of 0.1 unit/mg and was not activated significantly by the substrate, glucose 1-phosphate. Phosphorylation by protein kinase resulted in a 1300-fold activation. Glucose 6-phosphate inhibited the unphosphorylated enzyme more effectively than the phosphorylated form, and inhibition of the latter was cooperative. Glucose was a poor inhibitor for both the unphosphorylated and phosphorylated WT enzyme with Ki > 300 mM. The rate of phosphorylation by protein kinase depended on substrates and interactions of the amino terminus. Maltoheptaose increased the rate of phosphorylation of the WT enzyme by yeast phosphorylase kinase 5-fold. The 22-residue deletion mutant (Nd22) had overall kinetic properties similar to the WT enzyme, except that Nd22 was a better substrate for the protein kinase and the rate of phosphorylation was unaffected by maltoheptaose. The 42-residue deletion mutant (Nd42), which lacks the phosphorylation site, was measurably active, although much less active than phosphorylated WT. Sedimentation equilibrium analysis indicated that the WT, Nd22, and Nd42 exist as tetramer, partially dissociated tetramer, and dimer, respectively. Phosphorylation of the WT and Nd22 converted both to dimer. The results indicated that the amino terminus affects quaternary structure and mediates activity regulation through conformational transition.
Highlights
§ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of California, San Francisco, CA 941430448
It is not understood why glucose is a poor inhibitor for the yeast enzyme because all residues involved in binding glucose in the muscle enzyme are conserved
The structural mechanism by which phosphorylation activates muscle enzyme cannot apply to the yeast enzyme because of differing structural contexts; phosphorylation occurs on Ser14 in muscle enzyme and on ThrϪ10 in yeast enzyme
Summary
Glc-1-P, ␣-D-glucose 1-phosphate; Glc6-P, ␣-D-glucose 6-phosphate; BisTris, 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-propane-1,3-diol; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; WT, wild-type. The crystal structure of the unphosphorylated yeast enzyme has revealed that the amino-terminal extension binds near the catalytic site of the neighboring subunit in the homodimer (Fig. 1), suggesting that it prevents the substrate from entering the active site [17]. Both observations are consistent with the notion that phosphorylation activates the yeast enzyme by displacing the amino terminus from the active site. The oligomerization states of the wild-type and mutant enzymes in the presence of different activators and inhibitors were determined by sedimentation equilibrium to establish the role of the dimer-tetramer equilibrium and the amino terminus in the regulatory mechanism of yeast phosphorylase
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