GT 35-Glycogen Phosphorylase

The mammalian (largely rabbit) muscle enzyme has been the most investigated X-ray structural work favours a GTB fold. It has control, covalent and allosteric, at several levels and these are usually discussed in terms of a modification of the Monod-Wyman-Changeux model, in which the individual polypeptide monomers can adopt either a non-catalytic T ( tense ) state or a catalytic R ( relaxed ) state, but mixed oligomers (e.g. TR in a dimer) do not occur. In the extensive structural studies, oligomers with mixed conformations have never been observed. 

Phosphorylase b, without Serl4 covalently phosphorylated, can be made catalytically active by the binding of AMP, an allosteric activator. Its binding pocket arises from contact with several different regions of the monomer and from across the monomer-monomer interface. However, even in the presence of AMP, catalytic activities are lower than phosphorylase a. 

The monomer has a M, of 97 kDa and it is generally thought that the active form of both phosphorylase a and b is the dimer. In the case of phosphorylase b, in the absence of allosteric effectors the equilibrium lies towards the dimer at accessible protein concentrations. Phosphorylation promotes association to the tetramer by generating surface which becomes the interface of the dimer of dimers.The tetramer of phosphorylase a (and presumably phosphorylase b) is inactive and ligands have only modest effects on the association-dissociation equilibria. 

The enzyme is also subject to allosteric inhibition. When glucose binds at the active site, it stabiles the T-state conformation of the enzyme. The T state is also stabilised by bi- or tricyclic aromatic compounds such as caffeine or flavins, which bind at the entrance to the active site tunnel,and by acylated p-glucopyranosylamine derivatives, which bind similarly to glucose, but more tightly. A third allosteric site, formed at the interface of two subunits and normally an internal pool of water molecules , has recently been discovered in rabbit muscle phosphorylase b and human liver phosphorylase a. Occupancy of this site freezes the enzyme in the T state and inhibitors with 10 M dissociation constants from the site are being investigated in the treatment of diabetes. 

There is little motion of the protein, the main movement being closure of a loop over the active site after binding phosphate and substrate analogue. There are indications that when an acceptor maltooligosaccharide and glucose-1-phosphate are both bound, the phosphate is forced down and in an unfavourable torsional angle however there is no indication of any direct covalent interaction between the nucleophilic phosphate and the phosphate attached to the pyridoxal moiety, which had been proposed on the basis on NMR studies on glycogen phosphorylase. There is also no indication of any covalent intermediate. Structurally, the S i mechanism is plausible whereas the doubledisplacement mechanism would create difficulties. 

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