Phosphorylase potato

For the hydrosilylation reaction various rhodium, platinum, and cobalt catalysts were employed. For the further chain extension the OH-functionalities were deprotected by KCN in methanol. The final step involved the enzymatic polymerization from the maltoheptaose-modified polystyrene using a-D-glucose-l-phosphalc dipotassium salt dihydrate in a citrate buffer (pH = 6.2) and potato phosphorylase (Scheme 59). The characterization of the block copolymers was problematic in the case of high amylose contents, due to the insolubility of the copolymers in THF. 

With the development of enzymatic polymerization in solution, also first accounts for SIP appeared. Loos et al. [350] reported on enzymatic surface polymerization of glucose-l-phosphate with potato phosphorylase as the catalyst resulting in oligo- or poly-(a,l- 4)-D-glucopyranose. As initiator sites, immobilized malto-heptaose was used. Enzymatic grafting of hexyloxyphenol onto chitosan is reported by Payne and coworkers [351]. 

His first work in the Laboratory of Biological Chemistry was on the purification and properties of potato phosphorylase, but he soon changed his subject to the study of amino-containing sugars. It was not well understood at that time whether there were impurities in the products of separation and purification of proteins. Consequently, Onodera made up his mind to devote his whole life to identifying the chemical nature of amino-containing sugars. 

Fig. 6 Priming activity of glucose and maltooligosaccharides in the enzymatic polymerization using potato phosphorylase and glucose-1-phosphate as monomer [124] - Reproduced by permission of Portland Press Ltd.

Pfannemtiller et al. showed that it is possible to obtain carbohydrate-containing amphiphiles with various alkyl chains via amide bond formation. For this, mal-tooligosaccharides were oxidized to the corresponding aldonic acid lactones, which could subsequently be coupled to alkylamines [128-136]. Such sugar-based surfactants are important industrial products with applications in cosmetics, medical applications etc. [137-139]. The authors were also able to extend the attached mal-tooligosaccharides by enzymatic polymerization using potato phosphorylase, which resulted in products with very interesting solution properties [140, 141]. 

A full series of star-, network- and comb-like hybrid structures with oligosaccharides were synthesized by Pfannemiiller and coworkers (see Fig. 7) and it was shown that the attached oligosaccharides can be extended via enzymatic polymerization using potato phosphorylase [128, 129, 136, 156, 157]. 

Arsenate similarly replaces phosphate in various phosphorolysis reactions, so that sucrose phosphorylase catalyzes the hydrolysis of sucrose in its presence (23), potato phosphorylase can hydrolyze amylose and amylopectin (24), nucleoside phosphorylase can hydrolyze inosine... 

Fig. 33. Experimentally observed molecular-weight dependence of (S2)z for amylose chains grafted onto glycogen (filled circles) and partially debranched amylopectin (open circles). Grafting was achieved by potato phosphorylase (15 rays for glycogen and 12000 per amylopectin molecule), and by muscle phosphorylase (400 rays per glycogen molecule)142,143)...

Formation of glycogen chains by glycogen muscle phosphorylase and starch amylose by potato phosphorylase. 

Willnecker, Jahnke and Buehner96 reported recently the preliminary results of X-ray crystallographic analysis of the potato type-L isozyme at 2.6 A resolution, and showed that the overall folding of the potato phosphorylase is essentially the same as that of the rabbit muscle enzyme (more similar to the 7 -form than to the T-form). Only small differences were found in the core of the subunit, although local structures on the surface including the loops deviated significantly, being consistent with the sequence differences with some minor insertions and deletions as compared with the rabbit muscle enzyme (see above). As... 

Day 1 Incubation of Starch and Pi with Potato Phosphorylase, Preparation of the IR-45 Column... 

Prepare a 500-ml filter flask to receive the potato extract by pouring 250 ml of F O into it and marking the position of the top of the fluid in the filtration reservoir with a felt-tipped pen. Pour out the F120. Suspend about 100 mg of phenylmercuric nitrate in a few milliliters of H20 and pour it into the filter flask. Phenylmercuric nitrate is added to inhibit other enzymes and to prevent bacterial growth during the incubation of potato phosphorylase with starch. 

Table 12-2 Analysis of Glucose-1-Phosphate Isolated from Potato Phosphorylase Reaction pmol of Reducing /xmol of Inorganic Equivalents/0.1 ml Phosphate/0.1 ml...

McCready, R. M., and Hassid, W. Z. (1957). Preparation of a-D-Glucose-l-Phosphate by Means of Potato Phosphorylase. Methods Bnzymol 3 137. 

Hanes, C. S. 1940. The reversible formation of starch from glucose-l-phosphate catalyzed by potato phosphorylase. Proc. R. Soc. Lond. Ser. B. 120, 174-208. 

Scheme 4S. Potato phosphorylase-catalyzed synthesis of 2-desoxy-D-glucose 1-phosphate 135...

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 effect of temperature on the kinetics of the enzyme-catalyzed reaction is further complicated by changes in the pK s of the ionizable groups at the active center of the enzyme, thus producing additional changes in V and Km- A study of the variation of these pK s with temperature yields information on the heats of ionization of the ionizable group, AHj, by use of the equation AHj = —2.303 RT .dpK/ dT. The value of AHj has been calculated for the ionizing groups of bacterial alp/io-amylase and potato phosphorylase. ... 

Studies of the variation of enzymic activity with pH have indicated that a-amino and imidazolium groups are important entities in the potato phosphorylase molecule, and this evidence has been further substantiated by the results of photo-oxidation and acetylation experiments. Rabbit-muscle phosphorylase a has been modified by photo-oxidation and alkylation, and the effect of both procedures was markedly decreased by the presence of D-glucosyl phosphate or glycogen this result indicates that the amino acid residues affected are at, or near, the active center. These acids may be histidine and cysteine, but other interpretations of the results are possible. 

From the variation, with temperature, of the equilibrium constant of the overall reaction, the heat of the reaction has been shown to depend on pH, and values of 1.9—2.2 Kcal./mole have been obtained. Heats of ionization of groups at the active center of potato phosphorylase have been calculated, and are shown in Table XX. 

Heats of Ionization of Active Groups in Potato Phosphorylase... 

A double-displacement mechanism is unlikely, for D-glucose is not found in a system containing potato phosphorylase, arsenate, and D-glucosyl phosphate. ... 

Amylose was also prepared via in vitro polymerization of D-glucosyl phosphate catalyzed by a potato phosphorylase (120). A large excess amount of Glc-l-P is required in this equilibrium-controlled reaction. 

It appears that phosphorus is related to the formation of starch in the plant. Thus Hanes has synthesized a linear polysaccharide from a-D-glucopyranosc 1-phosphate (Cori ester) through the action of potato phosphorylase. Dunlap and Beckmann and likewise Cori have found that the B-fraction activates this enzymic synthesis, while the A-frac-tion is inactive. It has not been established whether this effect is due to the branched character of the B-fraction or to the presence of phosphate in its structure. 

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