Enzyme-substrate systems

The other constant in the equation, is often used to compare enzymes. is the substrate concentration required to produce half the maximum velocity. Under certain conditions, is a measure of the affinity of the enzyme for its substrate. When comparing two eu2ymes, the one with the higher has a lower affinity for its substrate. The value is an intrinsic property of the enzyme-substrate system and cannot be altered by changing [S] or [E]. 

Enzyme preparations of three Pieris species each contained B-thioglucosidase activity, a fact previously reported (36), and each inhibited hydrolysis in one or more combinations with a plant enzyme-substrate system. These data are being quantified and extended to include insect/preferred host-plant pairs. 

Drawing on this and the reported specificity of tannin-protein interactions ( ) leads to the conclusion that any useful in vitro modelling of the impact of tannins on digestion must consider more than pH and the concentrations of the buffer, enzyme, substrate, and tannin. The actual enzyme-substrate system must be nutritionally realistic to control for specificities of the reaction of tannins with proteins (including enzymes) gastrointestinal mucoproteins should perhaps also be included on the same grounds. Besides all this, misleading results nay still be obtained if bile surfactants are omitted from the equation. 

For an enzyme-substrate system obeying the simple Michaelis Menten mechanism, the rate of product formation when the substrate concentration is very large, has the limiting value 0.02 mol dmJ. At a substrate concentration of250 mg dnu, the rate is half this value, K/K assuming that K2 K j, calculated ... 

A foodstuff (or other sample) obtained by hydrolysis of a protein material is called a protein hydrolysate. The degree of hydrolysis measures the percentage of peptide bonds hydrolyzed during protein hydrolysis (Adler-Nis-sen, 1976). An advantage of the DH concept is that for a given enzyme/substrate system the DH is independent of five variables substrate concentration, enzyme/substrate ratio, pH. temperature, and time (Adler-Nissen, 1982). 

Fluidity can be assumed to stay constant when scaling up an enzyme-substrate system, provided that solutions of identical composition are used for the laboratory-scale model and the full-size plant design. Application of the design criterion in Eq. (19.36) assumes operation in the linear regime of transmembrane pressure AP up to about 1-2 bar, as described in Chapter, Section 8.5.1, Eq. (8.78), so that... 

Although the HRP/TMB system is usually a good, reliable, and sensitive combination, HRP has a number of alternative substrates, which can be used such as o-phenylene diamine. There are also number of options for the enzyme used other than HRP, such as alkaline phosphatase, which can be used in combination with the substrate p-nitrophenyl phosphate. It is important to note that if alkaline phosphatase is used, the wash buffer must not contain phosphate. Usually in this case a Tris-buffered rather than phosphate-buffered wash buffer is used. The choice of enzyme-substrate system depends on a number of factors, including price, sensitivity and whether a spectrophotometer filter is available for the substrate specific wavelength to be measured. 

Typical kinetic analyses of enzyme-substrate systems are based on observed rates of enzyme-catalyzed reactions over a range of enzyme and substrate concentrations. Analyses of this type are commonplace for soluble enzyme-soluble substrate systems in which measures of substrate and... 

After establishing a molecular bridge between antigen and the detection enzyme by means of specific antigen-antibody reactions, the antigen-antibody-enzyme complexes are visualized typically by chromogenic reactions. A variety of enzyme-substrate systems have been defined which yield soluble or insoluble, pigmented end products of different colors based on the needs of the researcher. 

The second approach involves directly computing the reaction coordinate for transformation of the enzyme-bound substrate(s) into product(s). Quantum mechanical treatments (see Section 2) are necessary to describe bond-making and breaking processes, however, and such methods are generally too expensive to apply to the whole enzyme-substrate system. Still, if this problem could somehow be circumvented (as has been attempted with QM/MM methods see below), then assumptions about the structures of species along the reaction coordinate could be avoided. 

Table 17-1 Soluble Colorimetric Enzyme/Substrate Systems for Detection of Antigens in the ELISA...

If we consider the back reaction Fj Fq + P. where k4 is not zero in the reactions of the enzyme-substrate system, modify the Michaelis-Menten kinetics. Show that when equilibrium is established, after a very long time, equilibrium concentrations of substrate and product are related by the following Haldane s relation... 

Computation of the reaction potential energy surface requires identification of reactant, TS, and product. Techniques that were discussed in Chapter 1 (and throughout the book) can be applied to enzyme/substrate systems too. Given the large number of atoms in proteins and, therefore, the large number of coordinates and gradients that must be minimized, other approaches are often more suitable. 

When contemplating performing a computation on an enzyme-substrate system, it is natural perhaps to shy away for fear of the huge size of the molecules and the impossibly long computational times. For this reason, most QM/MM computations of enzymes have employed a semiempirical method or more recently a density functional method for the QM region simply to keep the computational cost under control. Given the limitations of these methods, many of which have been described in previous chapters, there is rightful concern over the accuracy of computations performed with a practical implementation of the QM/MM procedure. 

The effect of the addition of methanol on the kinetics of an aqueous enzyme—substrate system has been studied for B. subtilis alpha-amylase acting on soluble starch. The Michaelis constant was found to be unaltered, but the rate of formation of products decreased as the concentration of methanol was increased. 

Table 6.2 Km Values for some enzyme- substrate systems...

Although static and dynamic disorder had been detected for other enzyme substrate systems before [10-12], one could argue that its observation in this case is an artifact of the way, how the experiments were performed. The nonspecific immobilization procedure might result in static disorder. Enzymes with different orientations on the surface might possess a different lid mobility and accessibility of the active site and, as a result, show different activities. And the use of the highly unnatural substrate might be a possible source of dynamic disorder. An alternative detection scheme for this class of enzymes, which solves these shortcomings, will be presented in Sect. 25.3. 

A good example of the range of parameters available from flow calorimetric data can be found from the study of enzyme/substrate systems. The kinetic nature of enzyme systems has been previously described by Michaelis and Menten. In the treatment discussed here, the parameters sought are the enthalpy, rate constant, Michaelis constant and the enzyme activity. The following example describes a study on the well-known enzyme substrate system, urea/urease. 

A computer simulation approach has been derived that allows detailed bimolecular reaction rate constant calculations in the presence of these and other complicating factors. In this approach, diffusional trajectories of reactants are computed by a Brownian dynamics procedure the rate constant is then obtained by a formal branching anaylsis that corrects for the truncation of certain long trajectories. The calculations also provide mechanistic information, e.g., on the steering of reactants into favorable configurations by electrostatic fields. The application of this approach to simple models of enzyme-substrate systems is described. 

Observed reaction kinetics for some enzyme substrate systems solubilized within a reversed micellar solution are enhanced relative to those observed In aqueous solution. This enhancement can be due to simple concentration of the reactants within the micelles, but can also be Influenced by the localization and orientation of the substrates being used. 

Bioluminescence is a unique type of chemiluminescence found in biological systems these reactions can be classified as either pyridine- or adenine-nucleotide linked systems or enzyme-substrate systems. In clinical enzymology, the most commonly used system is the firefly luciferin-luciferase system for the measurement of ATP ... 

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