Phosphorylase a and

Both phosphorylase a and phosphorylase kinase a are dephosphorylated and inactivated by protein phos-phatase-1. Protein phosphatase-1 is inhibited by a protein, inhibitor-1, which is active only after it has been phosphorylated by cAMP-dependent protein kinase. Thus, cAMP controls both the activation and inactivation of phosphorylase (Figure 18-6). Insulin reinforces this effect by inhibiting the activation of phosphorylase b. It does this indirectly by increasing uptake of glucose, leading to increased formation of glucose 6-phosphate, which is an inhibitor of phosphorylase kinase. 

In the 1940s Carl and Gertrude Cori isolated and purified an active form (phosphorylase a) and an inactive form (phosphorylase b) of an enzyme from muscle. Phosphorylase b is activated by AMP (see page 64). In 1955, Fischer Krebs found an enzyme that catalysed the conversion of phosphorylase b to phosphorylase a, together with hydrolysis of ATP to ADP. Thus it appeared to bring about phosphorylation of the enzyme. The enzyme was termed phosphorylase b kinase, was partially purified and the interconversion was established as... 

The principle underlying the changes in activity of a G-protein is similar to that of an interconversion cycle (Chapter 3). The classic example of an interconversion cycle is that between the two forms of the enzyme phos-phorylase phosphorylase a and b. The interconversions between b and a are catalysed by a protein kinase and a protein phosphatase. The similarities are as follows. 

The glucose 1-phosphate so formed can be used for ATP synthesis in muscle or converted to free glucose in the liver. Glycogen phosphorylase occurs in two forms the more active phosphorylase a and the less active phosphorylase b (Fig. 6-31). Phosphorylase a has two subunits, each with a specific Ser residue that is phosphorylated at its hydroxyl group. These serine phosphate residues are required for maximal activity of the enzyme. 

There are two forms of the enzyme, phosphorylase a and the less active form phosphorylase b. Phosphorylase a is an oligomeric protein with four major subunits. Phosphorylation of a serine hydroxy-group produces an active form of the enzyme. Removal of the phosphate causes a breakdown of the tetramer to a dimeric form which is the less active phosphorylase b. Re-activation is achieved by the enzyme phosphorylase kinase which catalyses the phosphorylation at the expense of ATP. 

Given that the only structural difference between phosphorylase a and phosphorylase b is that phos-phorylase a has a covalently bound phosphate on serine 14, do you expect phosphorylase a and phosphorylase b to elute as a single peak on DEAE-cellulose chromatography What if gel filtration was utilized ... 

Difference in charge allows the separation of phos-phorylase a from phosphorylase b with the use of DEAE-cellulose. Phosphorylase a and phosphorylase b should elute as a single peak upon gel filtration. 

Phosphorylase exists in two interchangeable forms active phosphorylase a and a normally inactive phosphorylase b. Phosphorylase b is a dimer and is converted into phosphorylase a by phosphorylation of a single serine residue on each subunit by the enzyme phosphorylase kinase. The process can be reversed and phosphorylase inactivated by removal of the phosphate group by protein phosphatase I (Fig. 2a) (see Topic C5). 

Fig. 7. Effects of different concentrations of glucagon on phosphorylase a and fructose 2,6-P2 levels in isolated rat hepatocytes. Reproduced from Ref. 112 by permission of the authors and publisher.

Phosphorylation of PhosbK by PKA yields a more active phospho-form of the enzyme (P-PhosbK) (with consequent generation of the more active phosphoenzyme phosphorylase a and increased breakdown of glycogen). Glycogen synthase (GS) phosphorylation yields the inactive P-GS form and hence inhibition of glycogen synthesis. Phosphorylation of adipocyte TGL yields the active P-TGL form with consequent increased breakdown in triglycerides to yield glycerol and fatty acids for export and catabolism. 

Rabbit-muscle phosphorylase a and b have been reported to dissociate into subunits having molecular weights of 242,000 and 135,000 respectively (see Table XVII), although it has been reported that subunits having weight 60,000 may be obtained on treatment with dodecyl sodium sulfate. Isozymes of rabbit-heart phosphorylase have, however, been reported, and these may be occasioned by differences in subunit structure. 

Comparison of the structures of phosphorylase a and phosphorylase b reveals that subtle structural changes at the subunit interfaces are transmitted to the active sites (see Figure 21.9). The transition from the T state (represented by phosphorylase b) to the R state (represented by phosphorylase a) entails a 10-degree rotation around the twofold axis of the dimer. Most importantly, this transition is associated with structural changes in a helices that move a loop out of the active site of each subunit. Thus, the T state is less active because the catalytic site is partly blocked. In the R state, the catalytic site is more accessible and a binding site for orthophosphate is well organized. 

Figure 21.9. Structures of Phosphorylase A and Phosphorylase B. Phosphorylase a is phosphorylated on serine 14 of each subunit. This modification favors the structure of the more active R state. One subunit is shown in white,...

All known actions of glucagon are mediated by protein kinases that are activated hy cyclic AMP. The activation of the cyclic AMP cascade results in a higher level of phosphorylase a activity and a lower level of glycogen synthase a activity. Glucagon s effect on this cascade is reinforced hy the diminished binding of glucose to phosphorylase a, which makes the enzyme less susceptible to the hydrolytic action of the phosphatase. Instead, the phosphatase remains hound to phosphorylase a, and so the synthase stays in the in-active phosphorylated form. Consequently, there is a rapid mobilization of glycogen. 

Effects on muscle enzymes. In addition to mentioned above 5 -lipoxigenase, tirosine hydroxilase and NO-synthase, some other enzymes are known to be affected by carnosine. Carnosine (as well as histidine) protects 3-phosphoglycerate dehydrogenase from heavy metals [6] and activates phosphorylase a and b (the former— in the acidic pH areas) [77], Stimulation of the over-all glycolytic process by the dipeptides is usually explained by their pH-buflfering capacity and protection of individual glycolytic enzymes from contaminations of heavy metals [78]. 

The dimeric skeletal-muscle phosphorylase exists in two interconvertible forms a usually active phosphorylase a and a usually inactive phosphorylase b (Figure 21.9). Each of these two forms exists in equilibrium between an active relaxed (R) state and a much less active tense (T) state, but the equilibrium for phosphorylase a favors the R state, whereas the ei rium for phosphorylase b favors the T state (Figure 21.10). Muscle phosphorylase b is active only in the presence of high concentrations... 

Figure 21.9 Structures of phosphorylase a and phosphorylase b. Phosphorylase a is phosphorylated on serine 14 of each subunit. This modification favors the structure of the more active R state. One subunit is shown in white, with helices and loops important for regulation shown in blue and red. The other subunit is shown in yellow, with the regulatory structures shown in orange and green. Phosphorylase b is not phosphorylated and exists predominantly in the T state. Notice that the catalytic sites are partly occluded in the Tstate. [Drawn from IGPA.pdb and INOJ.pdb.]...

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 original work by Sutherland and co-workers (Robinson et al., 1971)4 involved glucagon and epinephrine and their relationship to c-AMP and phosphorylase A and B... 

There is a cascade in which the cAMP-dependent protein kinase catalyzes a phosphorylation ofphosphorylase kinase. This activates phosphorylase kinase, which catalyzes the phosphorylation of phosphorylase from the inactive to the active form, which can then rapidly catalyze the mobilization of glycogen. The advantage of the cascade system is a multiplicative effect. That is, each cAMP-dependent protein kinase can catalyze the phosphorylation of a large number of phosphorylase kinases, which in turn can catalyze the phosphorylation of a large number of inactive phosphorylase molecules (i.e., phosphorylase b) to the active phosphorylated form (i.e., phosphorylase a), and each of these active phosphorylases can accelerate production of hexose phosphates from glycogen. This amplifies the stimulatory effect of cAMP and causes a more rapid mobilization of glycogen than would result from catalysis of a direct phosphorylation of phosphorylase by the cAMP-dependent protein kinase. 

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