Phosphorylases kinetics

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. 

In the majority of cases, fluorescent labels and probes, when studied in different liquid solvents, display single-exponential fluorescence decay kinetics. However, when they are bound to proteins, their emission exhibits more complicated, nonexponential character. Thus, two decay components were observed for the complex of 8-anilinonaphthalene-l-sulfonate (1,8-ANS) with phosphorylase(49) as well as for 5-diethylamino-l-naphthalenesulfonic acid (DNS)-labeled dehydrogenases.(50) Three decay components were determined for complexes of 1,8-ANS with low-density lipoproteins.1 51 1 On the basis of only the data on the kinetics of the fluorescence decay, the origin of these multiple decay components (whether they are associated with structural heterogeneity in the ground state or arise due to dynamic processes in the excited state) is difficult to ascertain. 

GLUCOSE TRACER KINETICS GLYCOGEN PHOSPHORYLASE GLYCOGEN SYNTHASE GLYCOLA.TE OXIDASE... 

ACTIN ASSEMBLY KINETICS POLYNUCLEOTIDE S -HYDROXYL-KINASE POLYNUCLEOTIDE PHOSPHORYLASE Polyol dehydrogenase,... 

Case Study Kinetic Crystallographic Studies of the Phosphorylase Enzyme. 

Kinetics. In a double-displacement mechanism the enzyme shuttles between free enzyme and the intermediate carrying the substrate fragment (here, the glycosyl enzyme). With sucrose phosphorylase the maximum velocity varies with the concentrations of sucrose and HP042 in the characteristic fashion expected for this "ping-pong" mechanism (Eq. 9-47)43... 

Earlier studies on the properties of phosphorylases isolated from various sources have indicated that their subunits are similar in size with about 100,000 daltons.15-17 The reaction proceeds in a rapid equilibrium random Bi-Bi mechanism as has been shown by kinetic studies with rabbit skeletal muscle phosphorylases a18-20 and b,21,22 rabbit liver enzyme,23 potato tuber enzyme,24 and the enzyme from E. coli.25) In contrast, the substrate specificities for various glucans differ considerably depending on the enzyme sources. The rabbit muscle enzyme has high affinity for branched glucans such as glycogen and amylopectin but low affinity for amylose and maltodextrin.26,27 The potato tuber enzyme can act on amylose, amylopectin, and maltodextrin but only poorly on glycogen,28,29 while the E. coli enzyme shows high affinity for maltodextrin.10 ... 

The purine nucleoside phosphorylases (PNPs) are N-ribosyltransferases (Figure 7-7) where transition state analogue design on the basis of kinetic isotope effects analysis has had success. The inhibition of phosphorylation catalyzed by human... 

Birck MR, Schramm VL (2004) Binding causes the remote [5 -3H]thymidine kinetic isotope effect in human thymidine phosphorylase. J. Am. Chem. Soc. 126 6882-6883... 

Kim, Y. K., Kitaoka, M., Krishnareddy, M., Mori, Y., and Hayashi, K. 2002. Kinetic studies of a recombinant cellobiose phosphorylase (CBP) of the Clostridium thermocellum YM4 strain expressed in Escherichia coli. J. Biochem., 132, 197-203. 

In this laboratory exercise, you will study the effects of phosphorylation and allosteric regulation on the activity of glycogen phosphorylase. In the first experiment, you will phosphorylate glycogen phosphorylase b in vitro using y-[32P]ATP and glycogen phosphorylase kinase. In the second experiment, you will study the effect of phosphorylation on glycogen phosphorylase activity, as well as the effect of AMP on glycogen phosphorylase b activity with the use of a coupled enzymatic (kinetic) assay (Fig. 15-3). 

The inhibitor constants, Kj, may be determined by measuring the variation of v with [S] at different concentrations of the inhibitor. Lineweaver—Burk plots can then be used to find K/ from measurements made in the presence, and absence, of inhibitor (see Fig. 2 and Table I). The kinetics of the inhibition of such enzymes as alpha-amylase, hefo-amylase, and phosphorylase have been studied, and inhibitor constants evaluated. 

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. ... 

Hydrogen-ion concentration effect on enzyme kinetics, 292, 327 on phosphorylase action, 355 mutarotation measurement by, 52... 

Phosphonomannans, fungal, 394,417 Phosphorus, in yeast mannan, 391 Phosphorylases active sites of, 346 amino acids of, 345 kinetics, 289,353 mechanism of action of, 356 properties of, 347... 

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