Phosphorylase reactions

Starch is stored in plant cells in the form of granules in the stroma of plas-tids (plant cell organelles) of two types chloroplasts, in which photosynthesis takes place, and amyloplasts, plastids that are specialized starch accumulation bodies. When starch is to be mobilized and used by the plant that stored it, it must be broken down into its component monosaccharides. Starch is split into its monosaccharide elements by stepwise phosphorolytic cleavage of glucose units, a reaction catalyzed by starch phosphorylase (Figure 7.23). This is formally an a(1 4)-glucan phosphorylase reaction, and at each step, the prod-... 

FIGURE 7.23 The starch phosphorylase reaction cleaves glucose residues from amy-lose, producing a-D-glucose-l-phosphate. 

The glycogen phosphorylase reaction degrades glycogen to produce limit dextrins, which are further degraded by debranching enzyme, as already described. 

The free-energy change of the glycogen phosphorylase reaction is AG° = +3.1 kj/mol. If [P ] = 1 mM, what is the concentration of glncose-1-P when this reaction is at equilibrium ... 

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. 

An enzymatic reaction intermediate formed by acylation of an acetal hydroxyl group. Such an intermediate is thought to occur in a number of reactions involving carbohydrates. The sucrose phosphorylase reaction is thought to proceed by way of an acyl-glucosyl intermediate. 

Table 4.6.18 Substrate mixture for the phosphorylase reaction. Instructions for the preparation of these solutions are given in section 4.6.21.3...

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.)... 

Glucose 1-phosphate, the end product of the glycogen phosphorylase reaction, is converted to glucose 6-phos-phate by phosphoglucomutase, which catalyzes the reversible reaction... 

All aminotransferases have the same prosthetic group and the same reaction mechanism. The prosthetic group is pyridoxal phosphate (PLP), the coenzyme form of pyridoxine, or vitamin B6. We encountered pyridoxal phosphate in Chapter 15, as a coenzyme in the glycogen phosphorylase reaction, but its role in that reaction is not representative of its usual coenzyme function. Its primary role in cells is in the metabolism of molecules with amino groups. 

Because the polynucleotide phosphorylase reaction does not use a template, the polymer it forms does not have a specific base sequence. The reaction proceeds equally well with any or all of the four nucleoside diphosphates, and the base composition of the resulting polymer reflects nothing more than the relative concentrations of the 5 -diphosphate substrates in the medium. 

The active sites of these enzymes can have a nitrogen ligand, usually as histidine (acid phosphatases and some protein phosphatases), a nucleophilic serine residue (alkaline phosphatases), a cysteine residue in which the thiol group can form a covalent species with the phosphate ester (protein phosphatases), or an aspartate-linked phosphate (plasma membrane ion pumps). The inhibitory form of vanadium is usually anionic vanadate V(V), but cationic vanadyl V(IV) has also shown strong inhibition of some types of phosphorylase reactions. Above neutral pH, speciation of vanadyl ions produces anionic V(IV) species capable of inhibition of enzymes in the traditional transition-state analogue manner [5],... 

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...

Construct a plot of A340 versus time for each of the 10 glycogen phosphorylase reactions (A-J). Determine the slope of the line for each plot (A 340/min). It may be helpful to construct four plots for tubes B-J one graph will contain the data for tubes B and C, another will contain the data for tubes D and E, another will... 

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. ... 

Fifty milliliters of the cell-free extract described above was fractionated by ammonium sulfate precipitation. The fraction precipitating between 30 and 50% saturation was redissolved in a total volume of 10 ml and dialyzed. The solution after dialysis occupied 12 ml and contained 30 mg protein/ml. Twenty microliters of the purified fraction catalyzed the phosphorylase reaction at a rate of 5.9 nmoles/min under the standard assay conditions. Calculate (a) the recovery of the enzyme and (b) the degree of purification obtained in the ammonium sulfate step. 

Since inorganic phosphate is indispensable for the nucleoside phosphorylase reaction, the phosphate concentration can be converted into an oxygen signal by using a nucleoside phosphorylase-xanthine oxidase sequence electrode (Watanabe et al., 1987 Watanabe, 1988). 2 mmol/1 of inosine has been shown to be necessary for optimal sensitivity to phosphate ion. The measuring range was 0.1-1 mmol/1 and the sensor could be used for 70 assays. 

Figure 5.3 Some phosphorylase reactions. The reactions as written are stoichiometric and thus independent of pH. However, both inorganic phosphate and glycosyl phosphates ionise over physiological pH ranges, so if the equilibrium constant is quoted in terms of total glycosyl phosphate and total inorganic phosphate, it is pH dependent.

Like the phosphorylase reaction in glycogen metabolism, although thiolysis is theoretically reversible, the equilibrium lies far over to the right in order for the cell to use it to build fatty acids it has to collect a formidably high concentration of acetyl-CoA and RCH2CO.SCoA. Again, a reaction possible in the test-tube is by no means so easy for the cell in practice. It is clear that all these facts, taken together, point to the existence of an alternative route of fatty acid synthesis. 

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