Glycogen phosphorylase

Regulation of glycogen phosphorylase in muscle is accomplished by many of the same enzymes that control glycogen synthesis. Phosphorylase kinase converts the dimeric phosphorylase from the inactive to the active form by Mg + and ATP-dependent phosphorylation of two identical serine residues. The principal enzyme that removes this phosphate may be protein phosphatase-1 (phosphorylase phosphatase). 

Whether the a- or a -subunit is present depends on the tissue. The subunits apparently differ in Ca + sensitivity, since the -subunit, discussed later, will not bind to the a -isozyme. 

A catalytic site on the y-subunit has considerable homology with the catalytic subunit of cAMP-dependent protein kinase (Chapter 30). Evidence for active sites on the a- and y8-subunits is weak. 

Phosphorylase kinase activity has an absolute requirement for Ca +, which binds to the 5-subunit. The amino acid sequence of this subunit is nearly identical to that of calmodulin, with four calcium binding sites, but unlike calmodulin, the 5-subunit is an integral part of the enzyme and does not dissociate from it in the absence of Ca +. In the presence of Ca +, kinase activity is further increased by phosphorylation of the a- and j6-subunits, catalyzed by cAMP-dependent protein kinase and several other kinases. Phosphorylation may activate the enzyme by disinhibiting 

Possible mechanism for regulation of glycogen metabolism in skeletal muscle by changes in cytosolic calcium. Increased glycogen breakdown may be coordinated with muscle contractions, as indicated here. The actual control scheme is probably more complicated, since phosphoprotein phosphatases are also involved. Interactions with cAMP-activated reactions, which also may complicate regulation, are not included. 

GP exists as a dimer composed of two identical subunits. Three GP isoenzymes are found in human tissues GP-LL, 

and GP-BB. Adult skeletal muscle contains only GP-MM. GP-LL is the predominant isoenzyme in liver and all other human tissues except for heart, skeletal muscle, and brain. GP-BB is the predominant isoenzyme in the human brain. In the heart, the isoenzymes BB and MM are found, but GP-BB is the predominant isoenzyme in the myocardium as well. 

In preliminary studies, GP-BB was significantly more sensitive than CK and CK-MB for AMI diagnosis during the first 3 to 4 hours after the onset of chest pain. Therefore GP-BB may be an important marker for the early diagnosis of AMI. Similar to other cytoplasmic proteins, such as myoglobin and CK-MB, the time course of GP-BB can be notably influenced by early reperfusion of the infarct-related coronary artery, with a more rapid increase and earlier peak. GP-BB is, however, not a heart-specific protein and its specificity as a marker for myocardial damage is limited. 

Methods were described for the estimation of GP activity in serum on the enzymic determination of glucose-1-phosphate in a coupled assay system and for the electrophoretic separation of GP isoenzymes. More recently, an immu-noenzymometric assay for the measurement of the isoenzyme GP-BB was developed. The upper reference limit of this research assay was 7 pg/L. 

Enzymes in this category include alanine and aspartate aminotransferases, glutamate dehydrogenase (GLD), ATP, 5 -nucleotidase (NTP), y-glutamyl transferase (GGT), glutathione S-transferase (GST), and serum cholinesterase (CHE). The aminotransferases and ALP are widely used. They have long been mistakenly called, as a group, liver function tests. They are not, of course, but the habit persists. GGT is widely available in the United States and on automated analyzers. The others have not been adopted as widely. 

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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)... 

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... 

AMP and ATP are competitive. Like ATP, AMP affects the affinity of glycogen phosphorylase... 

Glycogen phosphorylase conforms to the Monod-Wyman-Changeux model of allosteric transitions, with the active form of the enzyme designated the R state and the inactive form denoted as the T state (Figure 15.17). Thus, AMP promotes the conversion to the active R state, whereas ATP, glucose-6-P, and caffeine favor conversion to the inactive T state. 

FIGURE 15.17 The mechanism of covalent modification and allosteric regnlation of glycogen phosphorylase. The T states are bine and the R states bine-green. 

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. 

FIGURE 15.19 The hormone-activated enzymatic cascade that leads to activation of glycogen phosphorylase. 

Dephosphorylation of glycogen phosphorylase is carried out by phospho-protein phosphatase 1. The action of phosphoprotein phosphatase 1 inactivates glycogen phosphorylase. 

The cAMP formed by adenylyl cyclase (Figure 15.20) does not persist because 5 -phosphodiesterase activity prevalent in cells hydrolyzes cAMP to give 5 -AMP. Caffeine inhibits 5 -phosphodi-esterase activity. Describe the effects on glycogen phosphorylase activity that arise as a consequence of drinking lots of caffeinated coffee. 

Enzymes have evolved such that their values (or A o.s values) for substrate(s) are roughly equal to the in vivo concentration(s) of the substrate (s). Assume that glycogen phosphorylase is assayed at [P[] = A o.s in the absence and presence of AMP or ATP. Estimate from Figure 15.15 the relative glycogen phosphorylase activity when (a) neither AMP or ATP is present, (b) AMP is present, and (c) ATP is present. 

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