Phosphorylase primer

Synthetic oligonucleotides may be used as "primers and be elongated stepwise with the aid of polynucleotide phosphorylase (PNPase) and nucleoside diphosphates. 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

NORMAL ERROR CURVE STATISTICS (A Primer STAPHYLOCOCCAL NUCLEASE STARCH PHOSPHORYLASE... 

Fig. 5 Catalytic mechanism of glycogen phosphorylases. The reaction scheme accounts for the reversibility of phosphorolysis of oligosaccharides (R) in the presence of orthophosphate (upper half) and primer-dependent synthesis in the presence of glucose-l-phosphate (lower half). PL enzyme-bound pyridoxal BH-f a general base contributed by the enzyme protein. Reprinted with permission from [109]. Copyright 1990 American Chemical Society...

One of the remarkable properties of phosphorylase is that it is unable to synthesize amylose unless a primer is added (poly- or oligomaltosaccharide) ... 

Whelan and Bailey were also able to clarify the polymerization mechanism of the enzymatic polymerization with phosphorylase [124], Their results showed that the polymerization follows a multichain scheme in contrast to a single-chain scheme that was also proposed by some authors. In the multichain polymerization scheme, the enzyme-substrate complex dissociates after every addition step, whereas in the single-chain scheme each enzyme continuously increases the length of a single primer chain without dissociation. 

The strict primer dependence of the glycogen phosphorylases makes them ideal candidates for the synthesis of hybrid structures of amylose with non-natural materials... 

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. 

Sivak. M. N., 1992. (1— 4)-a-D-Glucan synthesis by a chloroplastic phosphorylase isolated from spinach leaves is independent of added primer. Carbohyd. Res. 227, 241-255. 

Single addition reactions to primer oligonucleotides, catalyzed by polynucleotide phosphorylase (EC 2.7.7.8), have been reported using 2 (3 )-0-substituted nucleoside 5 -diphosphate, where, apparently, the 2 -substituent prevents further reaction at the free 3 -hydroxyl group.302 Almost quantitative yields have been obtained by use of... 

Potato phosphorylase has successfully been employed for the enzymatic synthesis of 2-deoxyglucosyl 1-phosphate 135 (Scheme 45) [252]. D-glucal 136 was transferred onto maltotetrose 137 in the presence of the phosphorylase and catalytic amounts of phosphate to afford a modified maltooligosaccharide 138 that contained 2-deoxy-D-arabino-hexopyranosyl residues [253]. Formation of 2-deoxyglucosyl phosphate 135 can be explained by reverse phosphorolysis of the oligosaccharide 138. When soluble starch was employed as primer and an equimolar amount of phosphate was added, 2-deoxyglucosyl phosphate 135 was obtained in 50% isolated yield on a preparative scale. 

The maximum velocity of a phosphorylase reaction depends on the temperature and the pH, but is only obtained when the enzyme is saturated with both substrates that is, polysaccharide and inorganic phosphate for degradation, or primer and D-glucosyl phosphate for synthesis. Activities at 30° vary from 2.8 to 14 X 10 moles of D-glucosyl phosphate used up per min. per 10 g, of protein. ... 

The s)tnthetic reaction of the putative primer chain, however, only will occur when the ratio of Pi to a-Glc-1-P is less than the equilibrium value, which is 10.8 at pH 5, 6.7 at pH 6, and 3.1 at pH 7.0 [116]. These ratios are not obtained in vivo, as the concentration of Pj is 20- to 40-fold higher than the concentration of a-Glc-1-P [117,118] so that the in vivo conditions greatly favor degradation by phosphorylase, rather than synthesis. 

Because a degraded starch chain is the product of the degradation reaction, the synthetic phosphorylase catalyzed reaction would require a starch chain or a maltodextrin chain to be a substrate in the synthetic reaction, as shown above. This was the origin for the primer required reaction for starch bios)uithesis from the non-reducing-end of the primer, and it has pretty much been retained for 60 years [119,120,121]. 

For the synthesis of glycogen-type polysaccharides from a-n-glucopyran-osyl phosphate, two enzymes are required. Phosphorylases, in presence of a suitable primer, synthesize linear chains of -( —> 4)-linked n-glucose residues these are then converted into a branched polysaccharide by a branching enzyme. ... 

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