Phosphorylases cofactor

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. 

Pyridoxal phosphate is a coenzyme for many enzymes involved in amino acid metabolism, especially in transamination and decarboxylation. It is also the cofactor of glycogen phosphorylase, where the phosphate group is catalytically important. In addition, vitamin Bg is important in steroid hormone action where it removes the hormone-receptor complex from DNA binding, terminating the action of the hormones. In vitamin Bg deficiency, this results in increased sensitivity to the actions of low concentrations of estrogens, androgens, cortisol, and vitamin D. 

Pyridoxal phosphate is an essential cofactor in the glycogen phosphorylase reaction its phosphate group acts as a general acid catalyst, promoting attack by Pj on the glycosidic bond. (This is an unusual role for this cofactor its more typical role is as a cofactor in amino acid metabolism see Fig. 18-6.)... 

Glycogen phosphorylase. While PLP is ideally designed to catalyze reactions of amino compounds it was surprising to find it as an essential cofactor for glycogen phosphorylase (Fig. 11-5). Tire PLP is linked as a Schiff base in the same way as in other PLP-depen-... 

The polypeptide of type-L isozyme is composed of 916 amino acid residues. There are two covalently modified amino adds about one-fourth of the amino-terminal threonines are blocked by an acetyl group, and a lysyl residue (Lys762) is linked to the cofactor pyridoxal-P. The partial amino-terminal acetylation appeared to be a natural feature of type-L isozyme, and both the blocked and unblocked forms of type-L phosphorylase may exist in potato tubers. 

Vitamin Be has a central role in the metabolism of amino acids in transaminase reactions (and hence the interconversion and catabolism of amino acids and the synthesis of nonessential amino acids), in decarboxylation to yield biologically active amines, and in a variety of elimination and replacement reactions. It is also the cofactor for glycogen phosphorylase and a variety of other enzymes. In addition, pyridoxal phosphate, the metabolically active vitamer, has a role in the modulation of steroid hormone action and the regulation of gene expression. 

Studies on the reactivation of apoglycogen phosphorylase with a variety of analogs of pyridoxal phosphate have shown that the catalytic moiety is the 5 -phosphate group - only analogs with a reversibly protonatable dianion in this position have any activity In the nonactivated form of phosphorylase b, the phosphate is monoprotonated (-OPO3H ) when the enzyme has been activated, either allosterically or by phosphorylation (phosphorylase a), it is dianionic (-OPOa ). A glutamate residue in the active site acts as the proton acceptor or donor for this transition between the inactive and active forms of the cofactor. 

In addition, many examples of binding systems were investigated by stopped-flow fluorescence spectroscopy, such as the binding of calmodulin to calcineuiin, the binding of guanine to calf spleen purine nucleoside phosphorylase, the nucleotide cofactor binding to the Escherichia coli PriA Helicase, etc. 

Cofactors can be subdivided into two groups metals and small organic molecules (Table 8.2). The enzyme carbonic anhydrase, for example, requires Zn2+ for its activity (Section 9.2.1). Glycogen phosphorylase (Section 21,1.5). which mobilizes glycogen for energy, requires the small organic molecule pyridoxal phosphate (PLP). 

It acts as a cofactor for glycogen phosphorylase in glycogenolysis. Decreased glucose tolerance may be associated with vitamin B-6 deficiency. 

Endo- and exonucleases have been used successfully with nucleic acids and their analogs for organic synthetic purposes. For example, ATP was synthesized from AMP for use in cofactor recycling (Table 13-9, entry 1). The AMP was obtained from yeast RNA by cleavage with the nuclease Pi yielding a mixture of nucleoside monophosphates11011. In another report1731, nucleoside diphosphates were obtained by hydrolysis of RNA with nuclease Pi and a polynucleotide phosphorylase (the diphosphates are preferred because the diphosphates were more easily transformed to the nucleoside triphosphates than the monophosphates). 

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