Invertase kinetics

Bell et al. (2002) investigated the relationship between water mobility as measured by oxygen-17 NMR (transverse relaxation rate obtained from linewidth at half-height) and chemical stability in glassy and rubbery polyvinylpyrrolidone (PVP) systems. Reported results suggest that water mobility in PVP model systems was not related to Tg. The study did not find a link between water mobility and reaction kinetics data (half-lives) for degradation of aspartame, loss of thiamin and glycine, and stability of invertase. 

Applications of chemical kinetics to enzyme-catalyzed reactions soon followed. Because of the ease with which its progress could be monitored polarimetrically, enzyme hydrolysis of sucrose by invertase was a popular system for study. O Sullivan and Tompson (1890) concluded that the reaction obeyed the Law of Mass Action and in a paper entitled, Invertase A Contribution to the History of an Enzyme or Unorganized Ferment , they wrote [Enzymes] possess a life function without life. Is there anything [in their actions] which can be distinguished from ordinary chemical action ... 

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... 

Umbelliferose is most probably utilized in vivo through the primary attack of an a-D-galactosidase that would yield sucrose. It has been shown that the oligosaccharide is extremely resistant to hydrolysis by invertase.84 The kinetics of the inhibition showed94 that invertase. does not complex with umbelliferose the inhibition is probably attribu-... 

T. Kobayashi and M. Moo-Young, Kinetic and mass transfer behavior of immobilized invertase on ion-exchange resin beads, Biotechnol. Bioeng. 1973, 15, 47-67. 

Michaelis, L. and Menten, M.L. (1913) Kinetics of invertase action. Biochem. Zeit., 49, 333. 

Commercial yeast invertase (Bioinvert ) was immobilized by adsorption on anion-exchange resins, collectively named Dowex (1x8 50-400,1x4 50-400, and 1x2 100-400). Optimal binding was obtained at pH 5.5 and 32°C. Among different polystyrene beads, the complex Dowex-1x4-200/invertase showed a yield coupling and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrose hydrolysis for both soluble and insoluble enzyme were evaluated. The complex Dowex/inver-tase was stable without any desorption of enzyme from the support during the reaction, and it had thermodynamic parameters equal to the soluble form. The stability against pH presented by the soluble invertase was between 4.0 and 5.0, whereas for insoluble enzyme it was between 5.0 and 6.0. In both cases, the optimal pH values were found in the range of the stability interval. The Km and Vmax for the immobilized invertase were 38.2 mM and 0.0489 U/mL, and for the soluble enzyme were 40.3 mM and 0.0320 U/mL. 

A veiy important aspect of chemical kinetics is that dealing with the rates of enzyme-catalyzed reactions. Enzymes are a class of proteins that catalyze virtually all biochemical reactions. In this experiment we shall study the inversion of sucrose, as catalyzed by the enzyme invertase (/3-fractofuranidase) derived from yeast. The rate of the enzyme-catalyzed reaction will then be compared to that of the same reaction catalyzed by hydrogen ions. 

P8C-3 In an article on the kinetics of sucrose inversion by invertase with multiple steady sates in a CSTR [Chem. Eng. Common., 6, 151 (1980)], consider the following challenges Are the equations for Jf and K correct If not, what are the correct equations for these variables Can an analysis be applied to this. system to deduce regions of multiple steady statra ... 

Enzyme thermistors have also found applications in more research-related topics, such as the direct estimation of the intrinsic kinetics of immobilized bio-catalysts [64]. Here, the enzyme thermistor offered a rapid and direct method for the determination of kinetic constants (K , Km and Vm) for immobilized enzymes. For the system being investigated, saccharose and immobilized invertase, the results obtained with the enzyme thermistor and with an independent differential reactor system were in very good correlation, within a flow-rate range of 1 to 1.5 ml/min. 

Stefuca et al. (1990) proposed an ET method offering a rapid, convenient, and general approach to determine kinetic constants of immobilized biocatalysts. Here, a differential reactor (DR) was used for the measurement of the initial reaction rate of sucrose hydrolysis (Vallat et al. 1986). The enzyme column of the ET has been considered as a differential packed-bed reactor, and with a mathematical model, intrinsic kinetic constants of immobilized invertase were calculated from experimental DR and ET data. 

Stefuca V, Gemeiner P, Kurillova L, Danielsson B, Bales V (1990) Application of the enzyme thermistor to the direct estimation of intrinsic kinetics using the saccharose-immobilized invertase system. Enzyme Microb Technol 12 830 - 835 Stefuca V, Welwardova A, Gemeiner P, Jakubova A (1994) Application of enzyme flow microcalorimetry to the study of microkinetic properties of immobilized biocatalyst. Biotech-nolTech 8 497-502... 

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]...

This approach was used for the study of the kinetic properties of invertase immobilized on the cellulose bead surface [27,30,33]. In the following example the kinetic model described for invertase was used [34]... 

The significance of Eq. (28) is that the thermometric data can be used for evaluation of the kinetic parameters Km and K , and, thus, for rapid determination of the kinetic properties of IMB preparations. This had been performed in the previous work by investigating the kinetic properties of immobilized invertase [27]. Typical kinetic data from this study are presented in Fig. 7. In this... 

Fig. 8. Flow microcalorimetric investigation of kinetic properties of invertase bound to Eupergit C activated by concanavalin A. The line corresponds to calculated data. The model involving high substrate conversion was used and parameters were estimated by nonlinear regression Vm= 1950 mM min-1, Km= 3420 mM, K = 246 mM, P = 2.7 K (unpubl. results)...

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. 

Since quantities in the first parenthesis are constant, they can be regrouped in a single parameter, a. Thus, all parameters in the denominator of the kinetic term can be determined directly by nonlinear regression from thermometric data. Combining Eq. (39) with the kinetic model of the sucrose hydrolysis catalyzed by invertase represented by Eq. (25) we obtain... 

The last equation was used in several papers studying the kinetic properties of invertase, where substrate inhibition was evaluated in terms of the inhibition parameter K [27,30,31]. 

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... 

Fig. 78. Determination of sucrose with immobilized invertase and a GOD electrode, (a) Measuring cell, (b) Measuring curves of successive assay of glucose and sucrose. 1 current indication (/), 2 kinetic indication (d//df).

Yeast invertase,30 32 33 acid phosphatase,29 32 35 37 39 urease,29 /3-glucosi-dase,29 dCMP-amino hydrolase31 and malic enzyme34 36 have been immobilized in gel form on both flat and capillary membranes. Cellulosic and polyamide polymers have been used as supporting membrane matrices. In all instances, immobilized enzymes behave in a manner almost identical to their behavior in homogenous solution, independent of the nature of the polymer. Neither allosteric nor pseudo-allosteric enzymes, proteins whose kinetic behavior is affected by the presence of particular compounds in the reaction environment (ligands), show different kinetic behavior, as they do when subjected to less gentle immobilization procedures.31 34 36... 

Sucrose (common table sugar) is hydrolyzed to glucose and fructose (Section 16.3) in a classic experiment in kinetics. The reaction is catalyzed by the enzyme invertase. Using the following data, determine, by the Lineweaver-Burk method, whether the inhibition of this reaction by 2 M urea is competitive or noncompetitive. 

Since sucrose synthase was used to synthesize 1 -deoxy-T-fluorosucrose (1 -FS), it must also catalyze the breakdown of I -FS. The work of Guthrie et. al (14 utilizing modified methyl fructofuranosides as invertase substrates, showed that hydrogen bonding (OH donating) by the 1-OH is a requirement for substrate recognition. Sucrose and 1 -FS should therefore be differentially metabolized by the two enzymes. If the appropriate kinetic parameters are known to sufficient accuracy, the relative metabolic rates for the two substrates... 

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. 

Similar experiments done with invertase hydrolysis of the two substrates at ratios appropriate to allow analysis of the product, in spite of the large difference in reaction rates gives a ratio of about 4200 for the same kinetic parameters. 

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