Mechanism of Regulation in Yeast Glycogen Phosphorylase (original) (raw)
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Biochemistry-moscow, 2002
The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, α, of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One fragment, corresponding to residues 17-484 (PhbN"), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb. Truncation fragments of the α subunit were screened for interactions against these two substrate fragments. PhbC was not found to interact with any α constructs; however, PhbN" interacted with a region of α (residues 864-1014) that is near the phosphorylatable region of that subunit. PhbN" was also screened for interactions against a variety of fragments of the catalytic γ subunit of PhK; however, no interactions were detected, even with fulllength γ. Our results support the idea that amino acid residues proximal to the convertible serine of Phb are important for its specific interaction with the catalytic subunit of PhK, but that regions distinct from the convertible serine residue of Phb and from the catalytic domain of PhK may also be involved in the interaction of these two proteins.
Distinct phosphorylation signals converge at the catalytic center in glycogen phosphorylases
Structure, 1997
Background: Glycogen phosphorylases (GPs) catalyze the conversion of the storage form of carbohydrate (glycogen) to the readily usable form (glucose-1phosphate) to provide cellular energy. Members of this enzyme family have evolved diverse regulatory mechanisms that control a conserved catalytic function. The mammalian and yeast GPs are expressed as inactive forms requiring phosphorylation for activation. Phosphorylation of yeast GP occurs at a distinct site from that of mammalian GP. This work addresses the structural basis by which distinct activation signals relay to the conserved catalytic site in yeast and mammalian GPs. Such knowledge may help understand the principles by which diverse biological regulation evolves. Results: We have compared the crystal structures of the unphosphorylated and phosphorylated forms of yeast GP and propose a relay which links phosphorylation to enzyme activation. Structural components along the activation relay becomes more conserved within the GP family downstream along the relay, towards the catalytic center. Despite distinct upstream activation signals, a response element downstream of the relay leading to the catalytic center is conserved in all GPs. The response element consists of ten hydrophobic residues dispersed over two subunits of the homodimer. Phosphorylation induces hydrophobic condensation of these residues via structural rearrangement, which triggers conformation change of the active site GATE loop, leading to enzyme activation. Conclusions: Members of the GP family with diverse activation mechanisms have evolved from a constitutively active ancestral enzyme which has the TOWER hydrophobic response element in the active position. Diverse regulation evolved as a result of evolutionary constraint on the downstream response element in the active state, coupled with flexibility and variability in elements of the upstream relays.
Biochemical and biophysical research communications, 2017
The selective phosphorylation of glycogen phosphorylase (GP) by its only known kinase, phosphorylase kinase (PhK), keeps glycogen catabolism tightly regulated. In addition to the obligatory interaction between the catalytic γ subunit of PhK and the phosphorylatable region of GP, previous studies have suggested additional sites of interaction between this kinase and its protein substrate. Using short chemical crosslinkers, we have identified direct interactions of GP with the large regulatory α and β subunits of PhK. These newfound interactions were found to be sensitive to ligands that bind PhK.
Biochemistry. Biokhimii͡a, 2002
The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, alpha, of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One fragment, corresponding to residues 17-484 (PhbN'), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb. Truncation fragments of the alpha subunit were screened for interactions against these two substrate fragments. PhbC was not found to interact with any alpha constructs; however, PhbN' interacted with a region of alpha (residues 864-1014) that is near the phosphorylatable region of that subunit. PhbN' was also screened for interactions against a variety of fragments of the catalytic gamma subunit of PhK; however, no interactions were detected...
Role of protein phosphatase 2A in the control of glycogen metabolism in yeast
European Journal of …, 1995
The yeast homologues of mammalian protein phosphatase 2A (PP2A) are encoded by two genes, PPH21 and PPH22. To evaluate the role of these phosphatases in the control of glycogen metabolism, wild-type cells and mutants carrying deletions of PPH21 or PPH22 were studied. Our results indicate that the lack of a single gene product does not result in significant changes in glycogen content, glycogen synthase, and glycogen phosphorylase activities. Since the double disruption is very detrimental to the cell, the effect of lack of PP2A was evaluated by using strain H336, which carries a deletion of the PPH21 gene and has the PPH22 gene placed under the control of the GAL1 promoter, under conditions that allowed either progressive depletion or overexpression of PPH22. When grown on galactose, H336 cells contain 2-3-fold more PP2A activity than control cells. After 14 h in glucose, however, PP2A activity in strain H336 is markedly reduced. The decrease in PP2A activity correlates with a reduced accumulation of glycogen and a more pronounced inactivation of glycogen synthase while glycogen phosphorylase becomes more resistant to inactivation. These observations suggest a role for PP2A in controlling the activation states of both enzymes. The total amount of phosphorylase was also higher in the PP2A-depleted cells, as determined by both enzymic and immunochemical techniques. However, Northem-blot analysis revealed that this is not due to an increase in the phosphorylase mRNA, which is in fact reduced in these cells. In contrast, overexpression of PP2A causes an increased expression of glycogen phosphorylase and a resulting failure to accumulate glycogen. We conclude that PP2A is involved in regulating both the amounts and the activation states of glycogen synthase and glycogen phosphorylase.
Journal of Molecular Biology, 1995
Kinetic and crystallographic studies have characterized the effect of Research & Biotechnology 2-deoxy-glucose 6-phosphate on the catalytic and structural properties of The National Hellenic glycogen phosphorylase b. Previous work on the binding of glucose 6-phosphate, a potent physiological inhibitor of the enzyme, to T state Research Foundation, 48 Vas Constantinou Ave, Athens phosphorylase b in the crystal showed that the inhibitor binds at the 11635, Greece allosteric site and induces substantial conformational changes that affect the subunit-subunit interface. The hydrogen-bond from the O-2 hydroxyl of 2 Laboratory of Molecular glucose 6-phosphate to the main-chain oxygen of Val40' represents the only Biophysics, University of hydrogen bond from the sugar to the other subunit, and this interaction Oxford, Rex Richards appears important for promoting a more ''tensed'' structure than native T Building, South Parks Road state phosphorylase b. 2-Deoxy-glucose 6-phosphate acts competitively Oxford, OX1 3QU, UK with both the activator AMP and the substrate glucose 1-phosphate, with K i values of 0.53 mM and 1.23 mM, respectively. The binding of 3 School of Biology & 2-deoxy-glucose 6-phosphate to T state glycogen phosphorylase b in the Biochemistry, University of crystal, has been investigated and the complex phosphorylase b: Bath, Claverton Down 2-deoxy-glucose 6-phosphate has been refined to give a crystallographic R Bath, BA2 7AY, UK