Rabbit muscle phosphorylase

High resolution X-ray structures of the a- and b- form of rabbit muscle phosphorylase permit a view into some of the structural differences of the various allosteric forms of the enzyme. Furthermore, the data give an impression of the mechanism of binding of effectors and the influence that phosphorylation has on substrate binding and enzyme activity (Barford et al., 1991). The following discussion will be restricted to the observed consequences of phosphorylation. 

The phosphorylase-stimulation assay247 250 is based on the stimulation by branching enzyme of the unprimed reaction in absence of primer activity seen with rabbit muscle phosphorylase a activity. Branching enzyme present in the reaction mixture increases the number of non-reducing ends available to phosphorylase for elongation. 

Figure 6.1 also includes the sequence comparison with the rabbit muscle phosphorylase.I6,30) The sequence homologies of the animal enzyme with the potato type-L and type-H phosphorylase isozymes are 38% and 47%, respectively. Further sequence comparison of the plant and animal enzymes with the enzyme from E. co/i82 reveals an overall homology of at least 40%. These high similarities indicate that the phosphorylase family is one of the well-conserved protein groups. 

In conclusion, it should be pointed out that in marked contrast to the very extensive studies on rabbit muscle phosphorylase, little attention has been paid to enzymes from other sources. However, primary structures of plant phosphorylases have now been determined and bacterial expression systems for the plant enzymes have also been made available as reviewed in this article. We hope that future studies on the structure and function of plant phosphorylases without allosteric regulation and comparison with those of the highly regulated animal enzyme will provide valuable information on this interesting group of enzymes, phosphorylases. 

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. 

Other ions, or molecules, may be required for maximal activity of an enzyme, although the enzyme is active in their absence. Thus, mammalian alpha-amylases are activated by chloride ions, and rabbit-muscle phosphorylase a is activated by adenosine 5-phosphate. 

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. 

Studies of the variation of enzymic activity with pH have indicated that a-amino and imidazolium groups are important entities in the potato phosphorylase molecule, and this evidence has been further substantiated by the results of photo-oxidation and acetylation experiments. Rabbit-muscle phosphorylase a has been modified by photo-oxidation and alkylation, and the effect of both procedures was markedly decreased by the presence of D-glucosyl phosphate or glycogen this result indicates that the amino acid residues affected are at, or near, the active center. These acids may be histidine and cysteine, but other interpretations of the results are possible. 

The role of sulfhydryl groups in phosphorylase activity is not yet known. Modification with p-chloromercuribenzoate certainly causes loss of activity, but it is uncertain whether die sulfhydryl groups form a part of the active center, or are more important for the maintenance of the active conformation of the protein molecule. The second possibility seems the more likely, as p-chloromercuribenzoate causes rabbit-muscle phosphorylase to split up into subunits. Also, the inhibition by this reagent is not parallel to mercaptide formation, and adenosine 5 -phosphate (which causes changes in the conformation of the enzyme) protects against the inhibition. Unmodified sulfhydryl groups are believed to be important for the binding of adenosine 5 -phosphate. ... 

Such heavy metals as mercury, silver, and copper inhibit phos-phorylases, as does p-chloromercuribenzoate. Protamine inhibits rabbit-muscle phosphorylase, but not that from lobster. The potato enzyme is inhibited by some polyphenols, such as chlorogenic acid. ... 

Studies of the effect of temperature on V and have been made. From the variation of reaction velocity with temperature, activation energies have been found (see Table XVIII, p. 350). The activation energy of the removal of pyridoxal 5-phosphate from rabbit-muscle phosphorylase b has been calculated as 11.7 Kcal./mole, and the activation energy of its recombination with the enzyme was 22.3 Kcal./mole. ... 

Pyridoxal 5 -phosphate (PLP) was noticed to be a constituent of rabbit muscle phosphorylase in 1957, and since that time it has been shown that all a-glucan phosphorylases which give phosphorolysis products with retention of configuration contain PLP. The exact role of the PLP is still not known, though it has been shown that these a-glucan phosphorylases have an absolute requirement for PLP and that the Schiflfbase formed between PLP and glycogen phosphorylase can be reduced with borohydride without eliminating the catalytic activity of the enzyme. The P n.m.r. spectrum of PLP bound to phosphorylase b shows that deprotonation of the 5 -... 

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. 

Glycogen phosphorylase has been purified from adult A. suum muscle (3). The dephosphorylated enzyme, which requires AMP for activity, can be phosphorylated and activated by rabbit muscle phosphorylase kinase. Kinetic data and the observation that phosphorylase is not at equilibrium with its substrates and products, indicate that phosphorylase catalyzes the rate-limiting step of glycogenolysis in A. suum muscle. Phosphorylase activity correlates well with the rate of glycogenolysis observed in vivo... 

Chan, K.F.J. Graves, D.J. Isolation and physicochemical properties of active complexes of rabbit muscle phosphorylase kinase. J. Biol. Chem., 257, 5939-5947 (1982)... 

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 aim of this work is the comparison of two of the most commonly employed phosphorylases, potato phosphorylase and rabbit muscle phosphorylase b in the large scale production of LMWA. The decisive criterion should be the chain length distribution of the oligomers formed. 

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. 

Purified rabbit-liver phosphorylase phosphatase appears to be a single polypeptide chain having an approximate molecular weight of 3.4 x 10 it has an optimum pH of 6.9—7.4 and a Km for rabbit-muscle phosphorylase a of 2 p,M. The purification procedure is also applicable to rabbit-muscle phosphorylase... 

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