Phosphorylase b and

As early as 1938, it was known that glycogen phosphorylase existed in two forms the less active phosphorylase b and the more active phosphorylase a. In 1956, Edwin Krebs and Edmond Eischer reported that a converting enzyme could convert phosphorylase b to phosphorylase a. Three years later, Krebs and Eischer demonstrated that the conversion of phosphorylase b to phosphorylase a involved covalent phosphorylation, as in Eigure 15.17. 

This enzyme [EC 3.1.3.17] catalyzes the hydrolysis of phosphorylase a to produce two phosphorylase b and four orthophosphate. 

Fig. 8. A full white beam Laue diffraction pattern recorded from a crystal of glycogen phosphorylase b, and (P4(3)2(l)2 a = b = 128.8 A, c= 116.2 A) using a 0.2 mm diameter collimator the exposure time was 0.5 s...

Louise Johnson and her coworkers have determined the crystal structures of T and the R forms of muscle phosphorylase b and the R form of phosphorylase a. In parallel with this work, Robert Fletterick and coworkers determined the structure of the T form of muscle phosphorylase a. The crystal structures provide an incisive look at the structural changes that accompany the transitions from the T to the R conformation and from the nonphosphorylated form of the enzyme to the phosphorylated form. 

Rabbit muscle phosphorylase can exist in two forms an essentially inactive dimer, phosphorylase b, and an active tetramer, phosphorylase a. When AMP is noncovalently bound to phosphorylase b. or when phosphorylase b is phosphorylated at a serine residue by phosphorylase kinase, the enzyme is converted to the active, predominantly tetrameric, form (Chap. 11). The reaction can be reversed by the removal of AMP or the dephosphorylation of serines by phosphorylase phosphatase. 

Molecular weight markers of up to 205kDa (Sigma) were used with the tris-glycine buffer system, but it was possible to use crosslinked phosphorylase b and crosslinked bovine serum albumin markers (Sigma)... 

Figure 21.10 Phosphorylase regulation. Both phosphorylase b and phosphorylase a exist as equilibria between an active R state and a less-active T state. Phosphorylase b is usually inactive because the equilibrium favors the T state. Phosphorylase a is usually active because the equilibrium favors the R state. Regulatory structures are shown in blue and green.

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. 

Fig. 14 Typical time course of frequency changes of the amylopectin-immobilized 27-MHz QCM, responding to the addition of a the inactivated phosphorylase b and then AMP, and b the AMP-activated phosphorylase b in the presence of excess AMP. Immobilized amount of amylopectin = 620 20ngcm (6.2pmolcm ) on a QCM plate (4.9 mm Au electrode), [phosphorylase b] = 13.4 nM, [AMP] = 2mM 50 mM phosphate buffer, pH 6.8, 200 mM NaCl, 25 °C...

Several other purine nucleotide derivatives with alkyl halide substituents have appeared in the literature, including adenosine S -chloromethane phosphonate, adenosine 5 -chloromethylpyrophosphate, and adenosine 5 -()8-bromoethane phosphonate) (287-291). These compounds have been evaluated as affinity labels of such nucleotide-binding enzymes as leucyl- and tryptophanyl-tRNA synthetases (leucine- and tryptophan-tRNA ligases), phosphorylase b, and cAMP-... 

AMP is also an intermediate in de novo synthesis of ATP (reaction 3 below) and salvage synthesis of ATP (reactions 4, 5, and 8 below). AMP is an allosteric activator of glycogen phosphorylase b, and phosphofructokinase, as well as an allosteric inhibitor of fructose-1,6-bisphosphatase and adenylosuccinate synthetase. AMP is also an allosteric inhibitor of glutamine synthetase, an enzyme with a central role in nitrogen metabolism in the cell. 

Synthetic peptide variants of the sequence Ser-Asp-Gln-Glu-Lys-Arg-Lys-Gln-Ile-Ser-Val-Arg-Gly-Leu, which is found in phosphorylase b, have been used to reveal the structural features that are important for the interaction between rabbit-muscle phosphorylase b and phosphorylase b kinase. The results indicated that the important determinants in phosphorylase b recognized by phosphorylase b kinase reside in the linear sequence of amino-acids, particularly in the sequence Lys-Gln-Ile-Ser-Val-Arg. Although the precise role of these residues in the interaction with phosphorylase b kinase is not known, it appears that the... 

The changes in the quaternary structure of rabbit-muscle phosphorylase b at 4 °C have been followed by measuring the reactivity of the thiol group. A slow, concerted transition between the two dimeric forms was shown to take place, the rate of which is affected by adenosine monophosphate. A phos-phopeptide containing 14 residues, including the phosphorylated serine residue, derived from the amino-terminus of rabbit skeletal-muscle phosphorylase has been shown to induce the enzymic properties of phosphorylase a in phosphorylases b and b (a modified form in which the phosphorylated site has been removed by limited digestion with trypsin). Thus, these enzymes become partially active in the absence of adenosine monophosphate. 

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