Phosphorylases substrates

Nakayama C, Wataya Y, Meyer RB, Santi DV (1980) Thymidine phosphorylase. Substrate specificity for 5-substituted 2 -deoxyuridines. J Med Chem 23 962-964... 

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). 

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. 

Muscle Glycogen Phosphorylase Shows Cooperativity in Substrate Binding... 

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. 

The bromide (2a) reacted smoothly with 2,4-diethoxy-5-methylpyrimi-dine to give, after de-ethylation and deacylation, l-(2-deoxy-/ -D-arabino-hexopyranosyl)thymine (3) (15). The new nucleoside (3) is the first truly competitive inhibitor of a pyrimidine phosphorylase (7), that is, it inhibits the phosphorylase, yet is not a substrate for the enzyme. It was recently shown that 3 enhances the incorporation of 2 -deoxy-5-iodouridine in vivo in cats (8). 

Similarly, Ikehara, Tazawa, and Fukui (51) have found that the nucleotides 8-bromo and 8-oxoadenosine 5 -diphosphate, 8-bromo-, 8-oxo, and 8-dimethylaminoguanosine 5 -diphosphate are all inactive as substrates for homopolymer synthesis catalyzed by polynucleotide phosphorylase from Escherichia coli. Some of the results were later confirmed by Kapuler, Monny, and Michelson (52), who found that neither 8-bromo- nor 8-oxoguanosine 5 -diphosphate was active as a substrate for homopolymerization with polynucleotide phosphorylases isolated both irom Azotobacter vinelandii and . coli. 

They did find that these compounds behaved kinetically as competitive inhibitors of polymerization of the normal substrates e.g., guanosine 5 -diphosphate. These authors suggested that the successful completion of the polynucleotide phosphorylase reaction requires that the nucleotide be capable of assuming the anti conformation. Also, Kapuler and Reich (53) have found that both 8-bromo- and 8-oxoguanosine 5 -triphosphates are very poor substrates in the E. coli RNA polymerase reaction and are competitive inhibitors with respect to guanosine 5 -triphosphate as a substrate. 

The exact nature of levansucrase activity08 is not clear. It differs in certain respects from invertase, polymerase, fructosaccharase, and phosphorylase. Possibly the aldoside part of the substrate molecule is replaced by an enzyme-linked group, and partial decomposition of this levan precursor to aldose and ketose may furnish the energy necessary for levan synthesis. 

After removal of the phosphates by various phosphatases, the nucleosides are cleaved to the base by the same nucleoside phosphorylase that catalyzes the salvage reaction. The equilibrium constant for this reaction is near 1, so that it can go in either direction depending on the relative levels of the substrates and products. 

The remaining three types of CaMK are phosphorylase kinase, myosin light-chain kinase and CaMKIII. These each appear to phosphorylate fewer substrate proteins, and in some cases only one protein, under physiological conditions, and each may therefore mediate relatively fewer actions of Ca2+ in the nervous system. 

Sucrose phosphorylase can then be regarded as a rather versatile transglucosidase, capable of exchanging glycosidic and ester bonds and of donating D-glucose to a variety of substrates such as ketoses, an aldose, inorganic phosphate and arsenate. 

We may conclude from this discussion that in the formation of the phosphorylase-sucrose complex the glucosidic oxygen, the OH groups at C6, C4 and C2 of the glucopyranose moiety and the OH group at C3 of the fructofuranose moiety of the substrate are involved. 

Phosphorylase was studied in depth. The enzyme from muscle was different from that catalyzing the same reaction in liver. Muscle phosphorylase but not that from liver, was activated by AMP, an early example of enzyme regulation by a ligand which was not a substrate. [Allosteric regulation was not postulated until the work of Jacob and... 

The glucose concentration is the major factor regulating glycogen synthesis in liver. Glucose activates glucokinase directly as a substrate and indirectly via an increase in the concentration of fructose 6-phosphate. It also activates glycogen synthase but it inhibits glycogen phosphorylase (see text). 

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