Phosphorylase

Phosphorylase is an enzyme of wide, if not universal, occurrence in plants. It is also found in animal skeletal muscle, liver, and heart. In animals, phosphorylases usually exist in two forms, the a form and the b form. The b form is converted enzymically into the active a form by phosphorylation. Phosphorylases isolated from different sources appear to differ in structure, but it is not yet known whether they differ in action pattern. 

Crystalline enzymes have been obtained from rabbit, - human,pigeon, and lobster skeletal muscle, rabbit heart, and potatoes, and phosphorylases from dog heart, and dog, pig, and rabbit liver have been extensively purified. Preparative procedures have usually involved ammonium sulfate fractionation and heat treatment, but adsorption on calcium phosphate, 0-(2-diethyl-aminoethyl)cellulose chromatography, - and electrophoresis on 

The amino acid compositions of two phosphorylases have been determined, and are shown in Table XVI. The complete, amino acid sequence or three-dimensional structure of a phosphorylase is not yet known, and the task of delineating it will be difficult because of the large size of the molecules. However, the sequence of amino acids about an important phosphoserine residue has been reported for rabbit and human muscle phosphorylase a. In the rabbit enzyme, the sequence is 

Amino Acid Composition of Phosphorylase b of Babbit Muscle and Human Muscle 

Phosphorylases contain pjo idoxal 5-phosphate as prosthetic groups, and removal of the group causes a loss of activity, but this may be restored by incubation of the enzyme with an excess of pyridoxal 5-phosphate. The group is believed to be 

The most extensively used transferase in the field of polymer science is phosphorylase (systematic name (1— 4)-a-D-glucan phosphate a-D-glucosyltransferase EC 2.4.1.1). While this enzyme is responsible for the depolymerization of linear a-(l— 4) glycosidic chains in vivo it can also be used to synthesize linear a-(l— 4) glycosidic chains (amylose) in vitro. 

In vivo linear a-l,4-glucans are synthesized from adenosine diphosphate (ADP)-glucose by the enzyme glycogen synthase [16-19]. The enzyme as well as the monomer are quite sensitive and therefore most researchers (at least in the field of polymer science) prefer to use phosphorylase for the synthesis of amylose. 

Amylose is one component of starch which is the most abundant storage reserve carbohydrate in plants. Carbohydrates, such as starch, function as a reservoir of energy for later metabolic use. It is found in many different plant organs, including seeds, fruits, tubers and roots, where it is used as a source of energy during periods of dormancy and regrowth. 

Starch granules are composed of two types of a-glucan, amylose and amylo-pectin, which represent approximately 98-99% of the dry weight. The ratio of the two polysaccharides varies according to the botanical origin of the starch. 

In animals, a constant supply of glucose is essential for tissues such as the brain and red blood cells, which depend almost entirely on glucose as an energy source. 

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C. It is secreted along with noradrenaline by the adrenal medulla, from which it may be obtained. It may be synthesized from catechol. It is used as the acid tartrate in the treatment of allergic reactions and circulatory collapse. It is included in some local anaesthetic injections in order to constrict blood vessels locally and slow the disappearance of anaesthetic from the site of injection. Ultimately it induces cellular activation of phosphorylase which promotes catabolism of glycogen to glucose. 

Synthetic oligonucleotides may be used as "primers and be elongated stepwise with the aid of polynucleotide phosphorylase (PNPase) and nucleoside diphosphates. 

Showdomycin. Showdomycin (2-p-D-ribofuranosyhnaleimide) (7) is a maleimide C-nucleoside antibiotic synthesi2ed by S. showdoensis-, isoshowdomycin (8) and maleimycin (9) have also been isolated (1—6). Showdomycin is not phosphorylated by nucleoside kinase and is not a substrate for nucleoside phosphorylase. Once (7) enters the cell, it blocks the uptake of glucose and other nutrients. 

BVdU is degraded by thymidine phosphorylase more rapidly than the natural substrate, thymidine. This rapid enzymic degradation may present a problem in its clinical use. Moreover, herpes vimses develop resistance to BVdU, apparendy because of mutant vimses that have lower thymidine kinase activity. G. D. Seade has dropped further development of BVdU because of increased animal tumor incidence induced by prolonged dosing (1). 

FIGURE 6.28 Examples of protein domains with different numbers of layers of backbone strnctnre. (a) Cytochrome c with two layers of a-helix. (b) Domain 2 of phosphoglycerate kinase, composed of a /3-sheet layer between two layers of helix, three layers overall, (c) An nnnsnal five-layer strnctnre, domain 2 of glycogen phosphorylase, a /S-sheet layer sandwiched between four layers of a-helix. (d) The concentric layers of /S-sheet (inside) and a-helix (outside) in triose phosphate isomerase. Hydrophobic residnes are bnried between these concentric layers in the same manner as in the planar layers of the other proteins. The hydrophobic layers are shaded yellow. (Jane Richarelson)... 

Starch is stored in plant cells in the form of granules in the stroma of plas-tids (plant cell organelles) of two types chloroplasts, in which photosynthesis takes place, and amyloplasts, plastids that are specialized starch accumulation bodies. When starch is to be mobilized and used by the plant that stored it, it must be broken down into its component monosaccharides. Starch is split into its monosaccharide elements by stepwise phosphorolytic cleavage of glucose units, a reaction catalyzed by starch phosphorylase (Figure 7.23). This is formally an a(1 4)-glucan phosphorylase reaction, and at each step, the prod-... 

FIGURE 7.23 The starch phosphorylase reaction cleaves glucose residues from amy-lose, producing a-D-glucose-l-phosphate. 

Maltose phosphorylase cannot carry out a similar reaction. The P exchange reaction of sucrose phosphorylase is accounted for by a double-displacement mechanism where E = E-glucose ... 

Thus, in the presence of just Pi and glucose-l-phosphate, sucrose phosphorylase still catalyzes the second reaction and radioactive Pi is incorporated into glucose-l-phosphate over time. 

Maltose phosphorylase proceeds via a single-displacement reaction that necessarily requires the formation of a ternary maltose E Pi (or glucose E glucose-l-phosphate) complex for any reaction to occur. Exchange reactions are a characteristic of enzymes that obey double-displacement mechanisms at some point in their catalysis. 

B. Ca -calmodnlin (CaM)-dependent Phosphorylase kinase (PhK) —krk qis vrgl— phosphorylation by PKA... 

Glycogen Phosphorylase Allosteric Regulation and Covalent Modification 473... 

Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues, 97.44 kD). Each subunit contains a pyridoxal phosphate cofactor, covalently linked as a Schiff base to Lys °. Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface (Eigure 15.15). In addition, a regulatory phosphorylation site is located at Ser on each subunit. A glycogen-binding site on each subunit facilitates prior association of glycogen phosphorylase with its substrate and also exerts regulatory control on the enzymatic reaction. 

FIGURE 15.15 (a) The structure of a glycogen phosphorylase monomer, showing the locations of the catalytic site, the PLP cofactor site, the allosteric effector site, the glycogen storage site, the tower helix (residnes 262 throngh 278), and the snbnnit interface. 

Each subunit contributes a tower helix (residues 262 to 278) to the sub-unit-subunit contact interface in glycogen phosphorylase. In the phosphorylase dimer, the tower helices extend from their respective subunits and pack against each other in an antiparallel manner. 

Muscle Glycogen Phosphorylase Shows Cooperativity in Substrate Binding... 

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