Enzymes substrate reaction

Science is by no means incompatible with finding joy, meaning, and purpose in the world. 

One quick and popular immunoassay is the enzyme-linked immunosorbent assay, or ELISA (Kemeny, 1991). In ELISA, a surface is coated with antigen or antibody and dried. An antibody is then added to the test (see Section 6.20), usually suspended in water. Chemically linked to 

FIGURE 3.5.1 Michaelis-Menten reactions. (From Johnson, A.T., Biological Process Engineering An Analogical Approach to Fluid Flow, Heat Transfer, and Mass Transfer Applied to Biological Systems, John Wiley Sons, New York, 1999. With permission.) 

This assay usually takes several hours to complete, but there is interest in speeding it up. The time taken by the assay is composed of diffusion time, antigen-antibody binding (very quick), and enzyme-substrate kinetics of the Michaelis-Menten type (except that the substrate concentration does not limit the rate). Thus, a faster assay will come only as a result of minimizing each component of time (Johnson, 1999). 

To drive a reversible chemical reaction to one side, and produce the largest amount of products, greatly increase the concentration of reactants. 

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Enzyme-Catalyzed Reactions Enzymes are highly specific catalysts for biochemical reactions, with each enzyme showing a selectivity for a single reactant, or substrate. For example, acetylcholinesterase is an enzyme that catalyzes the decomposition of the neurotransmitter acetylcholine to choline and acetic acid. Many enzyme-substrate reactions follow a simple mechanism consisting of the initial formation of an enzyme-substrate complex, ES, which subsequently decomposes to form product, releasing the enzyme to react again. 

A combination of several rate constants affecting the rate of an enzyme-substrate reaction. 

Chemiluminescent labels, in which the luminescence is generated by a chemical oxidation step, and bioluminescent labels, where the energy for light emission is produced by an enzyme-substrate reaction, are additional labeling types (39,42). Luminol [521 -31 -3] CgHyN202, and acridine [260-94-6] C H N, derivatives are often used as chemiluminescent labels. 

Inhibitor An agent dial prevents die normal action of an enzyme without destroying it (e.g., a substance (inhibitor) B causes die slowdown of die enzyme-substrate reaction of A —> R). 

Chohnesterase-inhibiting pesticides (e g., organophosphate and carbamate pesticides) are detected by dipping the developed chromatogram in a solution of the enzyme chohnesterase followed by incubation for a short period. Then the plate is dipped in a substrate solution, e.g., 1-naphthyl acetate/fast blue salt B. In the presence of the active enzyme, 1-naphthyl acetate is hydrolyzed to 1-naphthol and acetic acid, and the 1-naphthol is coupled with fast blue salt B to form a violet-blue azo dye. The enzyme is inhibited by the pesticide zones, so the enzyme-substrate reaction does not occur pesticides are, therefore, detected as colorless zones on a violet-blue background [36]. 

Some ways to increase sensitivity include the use of MAbs or the use of a more sensitive substrate for the enzyme. The ELISA s sensitivity is from the inherent magnification of the enzyme-substrate reaction. 

EXAMPLE 2a SEARCH FOR A MECHANISM FOR THE ENZYME------------ SUBSTRATE REACTION... 

For the enzyme-substrate reaction of Example 2, the rate of disappearance of substrate is given by... 

Mechanism for enzyme catalyzed reactions. To explain the kinetics of enzyme-substrate reactions, Michaelis and Menten (1913) came up with the following mechanism, which uses an equilibrium assumption... 

When the presence of substance B causes the slowdown of the enzyme-substrate reaction of A to R, then B is called an inhibitor. We have various kinds of inhibitor action, the simplest models being called competitive and noncompetitive. We have competitive inhibition when A and B attack the same site on the enzyme. We have noncompetitive inhibition when B attacks a different site on the enzyme, but in doing so stops the action of A. In simple pictures this action is shown in Fig. 27.7. 

Given the Michaelis-Menten rate form to represent enzyme-substrate reactions (or catalyst-reactant reaction)... 

Yamazaki, I. Free radicals in enzyme-substrate reactions. In Free Radicals in Biology. Vol. II (Pryor, W. A., ed.). New York-San Francisco-London, Academic Press, 1977, pp. 183-218... 

Coupled enzyme systems have been used in enzymology to significantly increase the sensitivity of detection of enzyme-substrate reactions. The addition of coupled enzyme systems to an enzyme immunoassay should lead to several orders of magnitude increase in the sensitivity (48, 67). 

Suppose that the following sequence describes an enzyme-substrate reaction with product inhibition ... 

In the determination of steady state reaction kinetic constants of enzyme-substrate reactions, FABMS also provides some very unique capabilities. Since these studies are best performed in the absence of glycerol in the reaction mixture, the preferred method is that which analyzes aliquots which are removed from a batch reaction at timed intervals. Quantitation of the reactants and products of interest is essential. When using internal standards, generally, the closer in mass the ion of interest is to that of the internal standard, the better is the quantitative accuracy. Using these techniques in the determination of kinetic constants of trypsin with several peptide substrates, it was found that these constants could be easily measured (8). FABMS was used to follow the decrease in the reactant substrate and/or the increase in the products with time and with varying concentrations of substrate. Rates of reactions were calculated from these data for each of the several substrate concentrations used and from the Lineweaver-Burk plot, the values of Km and Vmax are obtained. 

Enzymes are catalysts, substances that play a role in specific chemical reactions, but which are not changed by the reaction. Thus, as a result of the enzyme-substrate reaction, the enzyme stays the same but the substrate is changed. Its shape can be different, or it can be broken down into products or combined with another molecule to make something new. 

Possible obstacles to using immunoassays are created by environmental factors, such as pH, temperature, or other food product ingredients, e.g., endogenous enzymes whose specificity is similar to the specificity of the enzyme present in the conjugate, colorings present in a given samples can interfere with the color obtained from the enzyme-substrate reaction, phenolic compounds which can adsorb proteins. 

N, A are the concentrations of excited and unexcited enzyme molecules, v, a are the corresponding excess concentrations i.e. the deviations from chemical equilibrium. S is the number of substrate molecules per unit volume, yo results from the long range interaction, Bv and aAav originate from the nonlinear enzyme-substrate reactions. Aside from "chemical" terms we have an additional "dielectric" term which consists of two parts a term describing the system s tendency to become ferroelectric, i.e. 

The principle upon which this approach is based is an enzyme-substrate reaction that produces a change in pH and a hydrolytically labile, pH-sensitive polymer containing dispersed therapeutic agent that can vary the erosion rate and concomitant drug release in response to that pH change. Figure 1 shows two types of drug... 

An alternative device is shown in Figure 1(b). In this device the therapeutic agent and the enzyme are both dispersed in the polymer, and the enzyme-substrate reaction occurs in the outer layers of the device. The altered pH in the outer layers then modifies erosion rate of the polymer. 

In developing such systems, we have investigated two enzyme-substrate reactions as a means of modulating polymer erosion rates ... 

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