Phosphorylase absence

The conversions of inosine to hypoxanthine (Fig. 25-17, step e), of guanosine to guanine (step g), and of other purine ribonucleosides and deoxyribonucleo-sides to free purine bases are catalyzed by purine nucleoside phosphorylase. " Absence of this enzyme also causes a severe immune deficiency which involves the T cells. However, B cell function is not impaired. ... 

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. 

Muscle phosphorylase is distinct from that of Hver. It is a dimer, each monomer containing 1 mol of pyridoxal phosphate (vitamin Bg). It is present in two forms phos-phoiylase a, which is phosphorylated and active in either the presence or absence of 5 -AMP (its allosteric modifier) and phosphorylase h, which is dephosphorylated and active only in the presence of 5 -AMP. This occurs during exercise when the level of 5 -AMP rises, providing, by this mechanism, fuel for the muscle. Phosphorylase a is the normal physiologically active form of the enzyme. 

TypeV Myophosphorylase deficiency, McArdle s syndrome Absence of muscle phosphorylase Diminished exercise tolerance muscles have abnormally high glycogen content (2.5-4.1%). Little or no lactate in blood after exercise. 

Fig. 8. The activity of muscle phosphorylase b (as a logarithm percentage of the activity of the fully AMP-activated enzyme in water) as a funaion of concentration (% v/v) of added organic cosolvent in the absence of AMP. It can be seen that long

Fig. 7.18. Regulation of glycogen metabolism in muscle. Phosphorylase kinase stands at the center of regulation of glycogen metabolism. Phosphorylase kinase may exist in an active, phosphorylated form and an inactive, unphosphorylated form. Phosphorylation of phosphorylase kinase is triggered by hormonal signals (e.g. adrenahne) and takes place via an activation of protein kinase A in the cAMP pathway. In the absence of hormonal stimulation, phosphorylase kinase can also be activated by an increase in cytosolic Ca. The active phosphorylase kinase stimulates glycogen degradation and inhibits glycogen synthesis, in that, on the one side, it activates glycogen phosphorylase by phosphorylation, and on the other side, it inactivates glycogen synthase by phosphorylation.

The rate of the reaction catalyzed by glycogen phosphorylase, as a function of the concentration of its main allosteric activator, AMR The curves shown in color were obtained in the presence of ATR Phosphorylase b (lower two curves) is almost completely inactive in the absence of AMR Its activity is half maximal at an AMP concentration of about 40 yuM. ATP greatly increases the concentration of AMP required for activity. Phosphorylase a (upper two curves) has about 80% of its maximal activity in the absence of AMP and reaches full activity at very low AMP concentrations it also is relatively insensitive to inhibition by ATP. 

The main effect of AMP on either phosphorylase b or phosphorylase a is to decrease the Km for P,. This change can be interpreted as we have interpreted the actions of allosteric effectors on phosphofructokinase and aspartate carbamoyl transferase, on the model that the enzyme can exist in two conformational states (R and T) with different affinities for the substrate. However, phosphorylase presents the additional complexity that the equilibrium constant (L) between the two conformational states can be altered by a covalent modification of the enzyme. In the absence of substrates, [T]/[R] appears to be greater than 3,000 in phosphorylase b but to decrease to about 10 in phosphorylase a. 

Figure 15. Rate of the debranching enzyme on phosphorylase limit dextrin in the presence of the irreversible inhibitor dimethyiarsenothioglucose in the presence and absence of a reversible inhibitor (Bis-Tris). Adapted from Gillard et al.

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. 

Phosphorylase kinase is one of the best characterized enzyme systems to illustrate the role of calcium ions in regulation of intermediary metabolism. Phosphorylase kinase is composed of four different subunits termed a (Mr 145000), /3 (MT 128000), y (A/r 45000) and 5 (Mr 17000) and has the structure (a/3y8)A [106]. Only one of its four subunits actually catalyses the phosphorylation reaction the other three subunits are regulatory and enable the enzyme complex to be activated both by calcium and cyclic AMP. The y subunit carries the catalytic activity the 8 subunit is the calcium binding protein calmodulin and is responsible for the calcium dependence of the enzyme. The a and /3 subunits are the targets for cyclic-AMP mediated regulation, both being phosphorylated by the cyclic-AMP dependent protein kinase. Calmodulin appears to interact with phosphorylase kinase in a different manner from other enzymes, since it is an integral component of the enzyme. Phosphorylase kinase has an absolute requirement for calcium, and is inactive in its absence. 

The inhibitor constants, Kj, may be determined by measuring the variation of v with [S] at different concentrations of the inhibitor. Lineweaver—Burk plots can then be used to find K/ from measurements made in the presence, and absence, of inhibitor (see Fig. 2 and Table I). The kinetics of the inhibition of such enzymes as alpha-amylase, hefo-amylase, and phosphorylase have been studied, and inhibitor constants evaluated. 

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. 

In resting muscle, nearly all the enzyme is in the inactive b form. When exercise commences, the elevated level of AMP leads to the activation of phosphorylase b. Exercise will also result in hormone release that generates the phosphorylated a form of the enzyme. The absence of glucose 6-phosphatase in muscle ensures that glucose 6-phosphate derived from glycogen remains within the cell for energy transformation. 

During exercise, [ATP] falls and [AMP] rises. Recall that AMP is an allosteric activator of glycogen phosphorylase b. Thus, even in the absence of covalent modification by phosphorylase kinase, glycogen is degraded. 

Incubation is carried out in the absence and presence of 5 -AMP. The ratio of the activity in the absence of 5 -AMP to the activity in the presence of 5 -AMP is termed phosphorylase activity ratio [165]. Increases in this ratio reflect relative increases in the percentage of phosphorylase in the activated form. Activity assayed in the presence of 5 -AMP is designated total phosphorylase activity. 

In the indirect method, crystalline phosphorylase a is incubated with the enzyme preparation to be assayed, aliquots are withdrawn, diluted (which stops the phosphatase reaction), and the diluted solutions are assayed in the absence of 5 -AMP for remaining phosphorylase a activity by incubation with glucose-l-phosphate and glycogen and determining the inorganic phosphate released. 

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