Muscle Pyridoxal Phosphate

Some 80% of the body s total vitamin Be is as pyridoxal phosphate in muscle, and some 80% of this is associated with glycogen phosphorylase. This does not seem to function as a reserve of the vitamin and is not released from the muscle in deficiency. 

Muscle pyridoxal phosphate is released into the circulation (as pyridoxal) in starvation as muscle glycogen reserves are exhausted and there is less requirement for glycogen phosphorylase activity. Under these conditions, it is potentially available for redistribution to other tissues, especially the liver and kidneys, to meet the increased requirement for gluconeogenesis from amino acids (Black et al., 1978). However, during both starvation and prolonged bed rest, there is a considerable increase in urinary excretion of 4-pyridoxic acid, suggesting that much of the vitamin Be released as a result of depletion of muscle glycogen and atrophy of muscle is not redistributed, but rather is ca-tabolized and excreted (Cobum et al., 1995). 

The normed muscle concentration of pyridoxal phosphate is of the order of 10 nmol per g in patients with McArdle s disease (glycogen storage disease from congenital lack of glycogen phosphorylase), the muscle content of pyridoxal phosphate is reduced to one-fifth of this. There is some evidence that patients with McArdle s disease show signs of vitamin Be deficiency, su esting that the muscle pool of the vitamin is important in maintenance of vitamin Be homeostasis (Beynon et ed., 1995). 

In most bacteria, pyridoxed phosphate is synthesized by condensation between 4-hydroxythreonine and the pentose sugar 1 -deoxy-xylulose phosphate, formed by condensation between pyruvate emd glyceraldehyde 3-phosphate. The details of the pathway are not known. In plants, fungi, emd some bacteria, the precursor is glutamine again, the pathway is unknown (Drewke and Leistner, 2001 Gupta et al., 2001). 

Decarboxylation aC—COOH Amino acid primary amine -i- CO2 

Loss of side chain aC—(3C Threonine aldolase Serine hydroxymethyltransferase 

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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. 

In general, pyridoxamine and pyridoxin are more stable than pyridoxal. All vitamers are relatively heat-stable in acid media, but heat labile in alkaline media. All forms of vitamin B6 are destroyed by UV light in both neutral and alkaline solution. The majority of vitamin B6 in the human body is stored in the form of pyridoxal phosphate in the muscle, bound to glycogen phos-phorylase. 

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. 

Six compounds have vitamin Bg activity (Figure 45-12) pyridoxine, pyridoxal, pyridoxamine, and their b -phosphates. The active coenzyme is pyridoxal 5 -phos-phate. Approximately 80% of the body s total vitamin Bg is present as pyridoxal phosphate in muscle, mostly associated with glycogen phosphorylase. This is not available in Bg deficiency but is released in starvation, when glycogen reserves become depleted, and is then available, especially in liver and kidney, to meet increased requirement for gluconeogenesis from amino acids. 

Shimomura, S., and Fukui, T. (1978) Characterization of the pyridoxal phosphate site in glycogen phos-phorylase b from rabbit muscle. Biochemistry 17, 5359. 

The answer is e. (Hardman, p 1563 ) The toxicity of INI I is mainly on the peripheral and central nervous systems (PNS, CNS). This is attributable to competition of 1NH with pyridoxal phosphate for apotryp-tophanase. This results in a relative deficiency of pyridoxine, which causes peripheral neuritis, insomnia, and muscle twitching among other effects. 

Muscle glycogen phosphorylase is one of the most well studied enzymes and was also one of the first enzymes discovered to be controlled by reversible phosphorylation (by E.G. Krebs and E. Fischer in 1956). Phosphorylase is also controlled allosterically by ATP, AMP, glucose and glucose-6-phosphate. Structurally, muscle glycogen phosphorylase is similar to its hepatic isoenzyme counterpart composed of identical subunits each with a molecular mass of approximately 110 kDa. To achieve full activity, the enzyme requires the binding of one molecule of pyridoxal phosphate, the active form of vitamin B6, to each subunit. 

Both muscle and liver have aminotransferases, which, unlike deaminases, do not release the amino groups as free ammonium ion. This class of enzymes transfers the amino group from one carbon skeleton (an amino acid) to another (usually a-ketoglutarate, a citric acid cycle intermediate). Pyridoxal phosphate (PLP) derived from vitamin is required to mediate the transfer. 

At least two distinct FDPases are found in animal tissues, one in liver and kidney, and the other in white muscle. The liver and kidney enzymes show minor differences in amino acid composition and in their response to agents, such as pyridoxal phosphate (4%), but these differences may be the result of modification during isolation (see above). On the other hand, the muscle enzyme is distinctly different in immunological properties as well as in amino acid composition (63, 74). All of the mammalian FDPases are similar in having a molecular weight of approximately 135,000, and all are composed of four subunits the... 

Although pyridoxine is taken up and phosphorylated by muscle (and other tissues), the resultant pyridoxine phosphate is not oxidized to pyridoxal phosphate. It has been suggested that the neurotoxicity of high intakes of pyridoxine (Section 9.9.6.4) may be caused by the uptake and trapping of pyridoxine, and hence competition with pyridoxal, resulting in depletion of tissue pyridoxal phosphate and a deficiency of the metabolically active form of the vitamin. 

Figure 5.63 Structures of ligands used to probe the role of pyridoxal phosphate in rabbit muscle glycogen phosphorylase.

Anai, M., Lai, C.Y., and Horecker, B.L. (1973) The pyridoxal phosphate-binding site of rabbit muscle aldolase. Arch. Bio-chem. Biophys. 156, 712-719. 

Muscle phosphorylase b consists of two protein subunits, each of which binds one molecule of pyridoxal phosphate. Optical activity can be demonstrated in the absorption band (near 333 nm) of pyridoxal phosphate by CD measurements, while in ORD spectra this Cotton effect is obscured by the protein (354). As measured by rotation in the ultraviolet region, the pyridoxal phosphate has little effect on the ordered structures of the protein (355). 

Fig. 38.5. Summary of the sources of NH4 for the urea cycle. All of the reactions are irreversible except glutamate dehydrogenase (GDH). Only the dehydratase reactions, which produce NH4 from serine and threonine, require pyridoxal phosphate as a cofactor. The reactions that are not shown occurring in the muscle or the gut can all occur in the liver, where the NH4 generated can be converted to urea. The purine nucleotide cycle of the brain and muscle is further described in Chapter 41.

This large, slow turnover pool is most likely in muscle, which contains about 70-80% of the vitamin B6 in the body (Cobum et al, 1985,1988b). The muscle pool is associated primarily with glycogen phosphorylase (Butler et al, 1985). Using pyridoxal phosphate as an indicator of glycogen phospho-... 

A third consequence is the question of whether glycogen phosphorylase conserves vitamin B6 because it has a very low rate of degradation or because it recycles the pyridoxal phosphate very efficiently. Based on the rapid decay of the free vitamin B6 pool in muscle, the slow turnover of the protein-bound vitamin B6 pool, the failure to detect apo-phosphorylase, and the agreement between turnover rates based on amino acid or vitamin B6 data, Beynon et al. (1986) concluded that recycling would be minimal. We feel that the effect of vitamin B6 intake on turnover rates tends to favor the recycling option under conditions of limited intake. However, more data are needed before a final decision can be made. 

Muscle phosphorylase exists in (a) and (b) forms. The latter is half the molecular weight of the former and requires AMP as an activator, but since the AMP concentration (5 x 10 m per liter) required for the activation is not reached in muscle, the (b) form is practically inactive. In passing from one form to another, two enzymes are involved—a proteolytic enzyme catalyzes the breakdown of phosphorylase (a), and another catalyzes the formation of (a) from (b) in the presence of ATP, magnesium, and manganese. The stoichiometry of the second reaction has been studied four molecules of ATP for each molecule of phosphorylase (b) appear to be involved. It has been shown that both phosphorylase (a) and (b) contain pyridoxal phosphate the (b) form contains two such molecules, and the (a) form contains four. The role of the coenzyme in the mechanism of action of phosphorylase is unknown, but the enzyme loses its activity when the coenzyme is dissociated from the main protein molecule by ammonium sulfate activity is restored by the addition of pyridoxal phosphate. 

The activation of phosphorylase is probably due to phosphorylation of the protein molecule rather than to dimerization. Phosphorylase (a) and (b) were isolated and crystallized from lobster muscle, and their sedimentation characteristics were found to be identical. Thus, in lobster muscle, the only difference between the active and inactive forms probably resides in the presence of phosphorus in the former. It was further observed that phosphorylase (a) contains one molecule of pyridoxal phosphate per 100,000 g of protein, and that, in contrast to (b), phosphorylase (a) did not require AMP for activity. A kinase that converts the (b) to the (a) form and a phosphatase that catalyzes the opposite reaction have also been purified from lobster muscle. Phosphorylase kinase has been purified from rabbit muscle. The enzyme is much more active if it is preincubated with ATP and magnesium prior to exposure to the substrate. The activity of the preincubated enzyme can be further stimulated by heparin, glycogen, and cyclic AMP. 

Again, muscle phosphorylase contains pyridoxal phosphate, and the pyridoxal is bound to the protein molecule by an imine group, although the formyl radical is not indispensable to phosphorylase activity. Reduction of the pyridoxal form of the phosphorylase with sodium borohydride does not inactivate the enzyme. 

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