Concentration kinetics invertase

In vitro Kinetic Parameters. Concentration kinetics for sucrose and 1 -FS hydrolysis by plant invertase are shown in Figure 3. The Km for sucrose hydrolysis was about 2 mM. Hydrolysis of 1 -FS was very slow at the concentrations tested, and the data are not sufficient to determine whether both Km and Vmax were changed by the substitution or if only a Km change has occurred. The data are consistent with a very high Km for 1 -FS as would be expected if the 1 -OH were required for substrate recognition by invertase. 

Further experiments by Brown and particularly Henri were made with invertase. At that time the pH of the reactions was not controlled, mutarotation did not proceed to completion, and it is no longer possible to identify how much enzyme was used (Segal, 1959). Nevertheless, in a critical review of kinetic studies with invertase, Henri concluded (1903) that the rate of reaction was proportional to the amount of enzyme. He also stated that the equilibrium of the enzyme-catalyzed reaction was unaffected by the presence of the catalyst, whose concentration remained unchanged even after 10 hours of activity. When the concentration of the substrate [S] was sufficiently high the velocity became independent of [S]. Henri derived an equation relating the observed initial velocity of the reaction, Vq, to the initial concentration of the substrate, [S0], the equilibrium constant for the formation of an enzyme-substrate complex, Ks, and the rate of formation of the products, ky... 

Fig. 6. Investigation of kinetic properties of immobilized invertase by flow microcalorimetry in the circulation mode. Initial sucrose concentration 51 mM, invertase immobilization by biospecific binding on concanavalin A-bead cellulose was prepared by binding on concana-valin A linked to chlorotriazine-activated cellulose, a Raw experimental thermometric data b data after conversion by the procedure indicated in Fig. 4. Concentrations were determined spectrophotometrically (open symbols) and by transformation of thermometric data explained in Section 5 (closed symbols) [32]...

The results of the experimental study and mathematical modeling of the invertase-catalyzed hydrolysis of sucrose are displayed in Fig. 8. The analysis of the output substrate concentration showed substantial substrate conversion. Therefore, the data were treated by differential equations (26) and (30), whereas the kinetic parameters were fitted using nonlinear regression. Regardless of the good agreement of the calculated and experimental values, it was concluded on the basis of a comparison of kinetic parameters obtained with those known from previous works on similar preparations of immobilized invertase [30] that this method did not provide reliable results. 

Inhibitors structurally related to the substrate may be bound to the enzyme active center and compete with the substrate (competitive inhibition). If the inhibitor is not only bound to the enzyme but also to the enzyme-substrate complex, the active center is usually deformed and its function is thus impaired in this case the substrate and the inhibitor do not compete with each other (noncompetitive inhibition). Competitive and noncompetitive inhibition effect the enzyme kinetics differently. A competitive inhibitor does not change but increases. Km (Fig. 25a) in contrast, noncompetitive inhibition results in an unchanged Km and an increased vmax (Fig. 25b). Some enzymes, e.g. invertase, are inhibited by high product concentration (product inhibition). 

With an excess of invertase and GOD in the enzyme membrane the total rate of sucrose determination is limited by the spontaneous mutarotation. Therefore the sensitivity towards sucrose is only about 10% of that for glucose (Scheller and Karsten, 1983). Kinetic (dl/dt) measurement even gives only 1% of the glucose signal at the same sucrose concentration. Application of coimmobilized mutarotase gives rise to an increase of the sensitivity by a factor of 6 for stationary measurement... 

Many of the early studies were conducted with enzymes from fermentation, particularly invertase, which catalyzes the hydrolysis of sucrose to monosaccharides D-glucose and D-fmctose. With the introduction of the concept of hydrogen ion concentration, expressed by the logarithmic scale of pH (Sorensen, 1909), Michaelis and Menten (1913) realized the necessity for carrying out definitive experiments with invertase. They controlled the pH of the reaction medium by using acetate buffer, allowed for the mutarotation of the product and measured initial reaction rates at different substrate concentrations. Michaelis and Menten described their experiments by a simple kinetic law which afforded a foundation for a subsequent rapid development of numerous kinetic models for enzyme-catalyzed reactions. Although the contribution of previous workers, especially Henri (1902, 1903), was substantial, Michaelis and Menten are regarded as the founders of modern enzyme kinetics due to the definitive nature of their experiments and the viability of their kinetic theory. 

In the development of the kinetic equations it was assumed that the concentration of the solvent (water) remains constant throughout the reaction. At high substrate concentrations this is not true, however, and there is a deviation from the theoretical equations. In the case of the inversion of sucrose by yeast invertase, the effect of the sucrose and water concentration was investigated by Nelson and Schubert (11), who found that the velocity of hydrolysis increases with substrate concentration to about 6 % sucrose but thereafter decreases steadily. As shown by these investigators, however, the decrease may be accounted for by the decrease in the water concentration as the sugar concentration increases. 

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