Coupled enzyme reaction

Table 3. Clinically Important Substances Detected Using Coupled Enzyme Reactions...

A wide variety of enzymes have been used in conjunction with electrochemical techniques. The only requirement is that an electroactive product is formed during the reaction, either from the substrate or as a cofactor (i.e. NADH). In most cases, the electroactive products detected have been oxygen, hydrogen peroxide, NADH, or ferri/ferrocyanide. Some workers have used the dye intermediates used in classical colorimetric methods because these dyes are typically also electroactive. Although an electroactive product must be formed, it does not necessarily have to arise directly from the enzyme reaction of interest. Several cases of coupling enzyme reactions to produce an electroactive product have been described. The ability to use several coupled enzyme reactions extends the possible use of electrochemical techniques to essentially any enzyme system. 

The final method of coupling enzyme reactions to electrochemistry is to immobilize an enzyme directly at the electrode surface. Enzyme electrodes provide the advantages already discussed for immobilization of enzymes. In addition, the transport of enzyme product from the enzyme active site to the electrode surface is greatly enhanced when the enzyme is very near to the electrode. The concept of combining an enzyme reaction with an amperometric probe should offer all of the advantages discussed earlier for ion-selective (potentiometric) electrodes with a much higher sensitivity. In addition, since the response of amperometric electrodes is linear, background can be selected. 

Microtiter plates HRP/H202/luminol AP/dioxetanes Firefly luciferin/luciferase Bacterial luciferin/luciferase Detection of enzymes and metabolites by direct or coupled enzyme reactions Determination of antioxidant and enzyme inhibitory activities Immunoassay... 

Yang and Schulz also formulated a treatment of coupled enzyme reaction kinetics that does not assume an irreversible first reaction. The validity of their theory is confirmed by a model system consisting of enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) with 2,4-decadienoyl coenzyme A as a substrate. Unlike the conventional theory, their approach was found to be indispensible for coupled enzyme systems characterized by a first reaction with a small equilibrium constant and/or wherein the coupling enzyme concentration is higher than that of the intermediate. Equations based on their theory can allow one to calculate steady-state velocities of coupled enzyme reactions and to predict the time course of coupled enzyme reactions during the pre-steady state. 

Creatine phosphokinase activity has been reported to be minimally inhibited by hemolysis. Hemoglobin concentrations of 1.25 g/100 ml inhibit 5% and 2.5 g/100 ml, 12% (N5). However, in methods utilizing adenosine diphosphate in the reaction mixture, hemolysates containing 100 mg of hemoglobin per 100 ml may have apparent activities of 5-100 units/liter. The activity is presumably related to adenylate kinase in the erythrocyte (S33). In methods utilizing adenosine diphosphate in a coupled enzyme reaction with hexokinase and glucose-6-phosphatase, the inhibitory effect can be eliminated by adding sufficient adenosine mono-... 

In a different approach, the hydrolase-catalyzed kinetic resolution of chiral acetates was studied using a high-throughput ee assay also based on an enzyme-coupled test, the presence of a fluorogenic moiety not being necessary [16]. The assay is based on the idea that the acetic acid formed by hydrolysis of a chiral acetate can be transformed stoichiometrically into NADH in a series of coupled enzyme reactions using commercially available enzyme kits (Fig. 9.10). The NADH is then... 

Y. Sekine and E.A.H. Hall, A lactulose sensor based on coupled enzyme reactions with a ring electrode fabricated from tetrathiafulvalen-tetra-cyanoquinodimetane, Biosens. Bioelectron., 13(9) (1998) 995-1005. 

The first step in developing an ELISA to detect the stem peptide product of the coupled enzyme reaction was to determine if antibodies to the stem peptide could be raised in animals. Only small quantities of stem peptide could be isolated or prepared enzymatically, so we used the commercially available pentapeptide portion of the stem peptide as the hapten in the hopes of generating high-affinity antibodies that would cross-react with the stem peptide. 

A competitive ELISA was developed to detect stem peptide generated from the coupled enzyme reaction as well as to measure the cross-reactivity of the antibody with small peptides. The ELISA format is pictured in Figure 4. BSA-pentapeptide (BSA-PP) was immobilized onto the wall of a microtiter plate. Samples or standards containing pentapeptide were then added, followed by rabbit anti-pentapeptide antibody. The amount of anti-pentapeptide antibody bound to immobilized BSA-PP was visualized by adding a second anti-rabbit IgG antibody... 

The effect of solvents such as DMSO, ethanol, and methanol, on the MurD coupled enzyme reaction and the ELISA was studied. The MurD enzyme reaction was run in the presence and absence of Mur enzymes to generate minimum and maximum signals. Final solvent concentrations up to 2% had no effect on the reaction when measured by either HPLC or in the ELISA. 

The interplay of mass transfer, partition and enzymatic substrate conversion determines the dynamic measuring range, response time, and accessibility towards interferences of enzyme sensors. New principles for designing the analytical performance by coupled enzyme reactions are presented in this paper ... 

The final method of coupling enzyme reactions to electrochemistry is to immobilize a biocatalytic material directly at the electrode surface. This biocatalytic material can be an immobilized enzyme, bacterial particles, or a tissue slice, as shown in Fig. 8. The biocatalyst converts substrate (analyte) into product, which is measured by the electrode. Electrodes of this type can be potentiometric or Faradaic, and are often referred to as biosensors. ... 

An ingenious method for detecting the transfer of the terminal phosphate of ATP in ATP-coupled enzyme reactions has been devised, If an enzyme becomes reversibly phosphorylated, and the /9-phosphate of the bound ADP is free to rotate, an isotopic label in the terminal oxygen bridge will become scrambled between three oxygen atoms (Scheme 2). In order to demonstrate the formation of a complex... 

Unfortunately, most enzymes do not obey simple Michaelis-Menten kinetics. Substrate and product inhibition, presence of more than one substrate and product, or coupled enzyme reactions in multi-enzyme systems require much more complicated rate equations. Gaseous or solid substrates or enzymes bound in immobilized cells need additional transport barriers to be taken into consideration. Instead of porous spherical particles, other geometries of catalyst particles can be apphed in stirred tanks, plug-flow reactors and others which need some modified treatment of diffusional restrictions and reaction technology. 

Determination of enzyme activity with a coupled enzyme reaction Sometimes an indicator reaction is necessary to establish... 

Of all types of biosensors, metabolism sensors based on the molecular analyte recognition and conversion have been most intensively studied. According to the degree of integration of the biocomponents they can be classified into monoenzyme sensors, biosensors using coupled enzyme reactions, organelle, microbial, and tissue-based sensors. The sequence of the following sections corresponds to this classification. 

Parallel coupling constitutes another type of coupled enzyme reactions. This includes the competition of two enzymes for a common substrate as well as the conversion of alternative substrates and the competitive binding of a substrate and an inhibitor to an enzyme. Thus, analytes become measurable even though they cannot be converted to readily detectable products. Coupled enzyme reactions can also be used to eliminate disturbances of the enzyme or transducer reaction caused by constituents of the sample. Compounds interfering with the signal transduction can be transformed into inert products by reacting them with an (eliminator) enzyme which can be coimmobilized with the analyte-converting (indicator) enzyme in the vicinity of the transducer. On the other hand, constituents of the sample which are at the same time intermediate products of coupled enzyme reactions and will thus... 

Fig. 140. Internal signal processing in biosensors using coupled enzyme reactions. (Redrawn from Scheller et al., 1985b).

Fig. 141. Coupling of molecular recognition and internal signal processing, e.g. amplification and chemical filtering, by coupled enzyme reactions in biosensors.

The third chapter concentrates on metabolism sensors, which are arranged according to the degree of biocatalyst integration. The various different ways of coupling enzymes with transducers in monoenzyme sensors are exemplified by the determination of glucose and urea. The current state of the art is shown for monoenzyme sensors for some further 25 analytes and classes of analytes. Coupled enzyme reactions are shown to provide expansion of the biosensor concept to new analytes and to multiparameter assays as well as to an improvement of such analytical parameters as specificity and sensitivity. This chapter offers for the first time a complete overview of the potentials of coupled enzyme reactions in biosensors. 

Glucose oxidase/peroxidase Couple enzyme reaction Glucose + HPR chromogen ... 

Another example of a coupled enzyme reaction demonstrates the versatility of the transaminase system in biocatalysis. Using a racemic d,L-amino acid mixture as the starting material, the enzyme D-amino acid oxidase from Trigonopsis mriabilis will convert the D-amino acid in the mixture selectively into the corresponding 2-keto acid. The L-amino acid of the d,l- pair is neither a substrate nor an inhibitor of d-amino acid oxidase. If a transaminase is present in the same reaction mixture, the 2-keto acid can be transaminated in the presence of L-aspartate to the corresponding L-amino acid. The entire reaction can be driven to completion as described previously by decarboxylation of the oxaloacetate. Thus, in a single pot, racemic d,l-amino acids can be convened directly into optically active L-amino acids (Fig. 12.7-11). 

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