Enzyme reactions, coupling

Similar complex oscillatory phenomena have been observed in a closely related model containing two regulated enzyme reactions coupled in a different manner (Li, Ding Xu, 1984). An additional indication of the generality of the results obtained in the multiply regulated biochemical system is given by the study of the model for the synthesis of cAMP in Dictyostelium amoebae. In addition to simple periodic oscillations and excitability (see chapter 5), this realistic model based on experimental observations also predicts the appearance of more complex oscillatory phenomena in the form of birhythmicity, bursting and chaos (chapter 6). 

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

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

The simple cases where one enzyme is employed afford a limited scope of potential targets. Usually two or more enzyme reactions are coupled, as exemplified by the development of a piezoelectricaHy-transduced biocatalytic biosensor that couples two enzyme reactions to detect glucose [492-62-6] ... 

Another quite general approach is to employ a coupled assay (Figure 7-10). Typically, a dehydrogenase whose substrate is the product of the enzyme of interest is added in catalytic excess. The rate of appearance or disappearance of NAD(P)H then depends on the rate of the enzyme reaction to which the dehydrogenase has been coupled. 

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. 

In this chapter, a novel interpretation of the membrane transport process elucidated based on a voltammetric concept and method is presented, and the important role of charge transfer reactions at aqueous-membrane interfaces in the membrane transport is emphasized [10,17,18]. Then, three respiration mimetic charge (ion or electron) transfer reactions observed by the present authors at the interface between an aqueous solution and an organic solution in the absence of any enzymes or proteins are introduced, and selective ion transfer reactions coupled with the electron transfer reactions are discussed [19-23]. The reaction processes of the charge transfer reactions and the energetic relations... 

A common characteristic of metabolic pathways is that the product of one enzyme in sequence is the substrate for the next enzyme and so forth. In vivo, biocatalysis takes place in compartmentalized cellular structure as highly organized particle and membrane systems. This allows control of enzyme-catalyzed reactions. Several multienzyme systems have been studied by many researchers. They consist essentially of membrane- [104] and matrix- [105,106] bound enzymes or coupled enzymes in low water media [107]. 

Figure 6.7 illustrates the voltammetric response of the third-generation SOD-based 02 biosensors with Cu, Zn-SOD confined onto cystein-modified Au electrode as an example. The presence of 02" in solution essentially increases both the cathodic and anodic peak currents of the SOD compared with its absence [150], Such a redox response was not observed at the bare Au or cysteine-modified Au electrodes in the presence of 02". The observed increase in the anodic and cathodic current response of the Cu, Zn-SOD/cysteine-modified Au electrode in the presence of 02 can be considered to result from the oxidation and reduction of 02, respectively, which are effectively mediated by the SOD confined on the electrode as shown in Scheme 3. Such a bi-directional electromediation (electrocatalysis) by the SOD/cysteine-modified Au electrode is essentially based on the inherent specificity of SOD for the dismutation of 02", i.e. SOD catalyzes both the reduction of 02 to H202 and the oxidation to 02 via a redox cycle of its Cu (II/I) complex moiety as well as the direct electron transfer of SOD realized at the cysteine-modified Au electrode. Thus, this coupling between the electrode and enzyme reactions of SOD could facilitate the development of the third-generation biosensor for 02". ...

Enzyme-coupled ECL enables the selective determination of many clinical analytes that are not in themselves directly electrochemiluminescent, but that can act as substrates for a variety of enzymic reactions. There are two general strategies for ECL the use of dehydrogenase enzymes, which convert NAD+ to NADH, and oxidase enzymes, which produce hydrogen peroxide. 

Studies have also been made on the coupling of the CL luminol-hydrogen peroxide in HTAB reversed micellar medium detection system with enzyme reactions [64], The use of HTAB reversed micellar medium permits the simultaneous performance of both reactions, enzyme and CL detection, at a mild pH in the... 

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... 

In the structurally coupled QM/MM implementation of Zhang et al. [55, 56], in which the QM/MM boundary was treated by use of the pseudobond approach [55, 57], the QM/MM minimization of the QM part is combined with FEP calculations. In this procedure the energy profile of the enzyme reaction is first determined by use of QM/MM energy minimizations. The structures and charges of the QM atoms are then used, in the same manner as in the QM/FE method, to determine the role of environment on the energy profile of the reaction. In this way the effects of a large number of MM conformations of protein and solvent environment can be included in the total energies. 

The subject of biochemical reactions is very broad, covering both cellular and enzymatic processes. While there are some similarities between enzyme kinetics and the kinetics of cell growth, cell-growth kinetics tend to be much more complex, and are subject to regulation by a wide variety of external agents. The enzymatic production of a species via enzymes in cells is inherently a complex, coupled process, affected by the activity of the enzyme, the quantity of the enzyme, and the quantity and viability of the available cells. In this chapter, we focus solely on the kinetics of enzyme reactions, without considering the source of the enzyme or other cellular processes. For our purpose, we consider the enzyme to be readily available in a relatively pure form, off the shelf, as many enzymes are. 

Coupled enzyme assays provide a good alternative to chemical modification and permit a kinetic technique to be employed. In a coupled assay, the rate at which the product is formed is measured by using the product of the reaction as a substrate for a second enzyme reaction which can be monitored more easily. Coupled assays offer great flexibility in enzyme methodology while still retaining all the advantages of continuous monitoring techniques and are illustrated by Procedure 8.5. 

Klinman, J.P. (1991). Hydrogen tunneling and coupled motion in enzyme reactions. In Enzyme Mechanism from Isotope Effects, Cook, P.F. (ed.), pp. 127-148. CRC Press, Boca Raton... 

The basic system considered in this study relies on well-dehned enzymic reactions and is designed to function as a node or biochemical neuron in biochemical networks. This system involves two enzyme-catalyzed reactions, coupled to one another by the use of a cofactor, the latter being cycled continuously between the two. In addition, the two consumable substrates are fed into the system continuously at predetermined concentrations and rates. Also considered in this work was an extension of the basic system termed the extended basic system. The extended system relies on the same reactions as those in the basic system in addition, an external compound, inhibitory to one of the enzymes, is fed into the system. 

Phase II reactions (conjugate formation). Type II reactions couple their substrates (bilirubin, steroid hormones, drugs, and products of phase I reactions) via ester or amide bonds to highly polar negatively charged molecules. The enzymes involved are transferases, and their products are known as conjugates. 

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