Coupling of enzyme reactions

From the analysis of the coupling of enzyme reaction and mass transfer the following conclusions may be drawn for the design of biosensors. 

Fig. 86. Coupling of enzyme reactions for the design of a sensor family based on LMO. CK = creatine kinase, PK = pyruvate kinase, MDH = malate dehydrogenase, PEP = phosphoenolpyruvate, ALAT = alanine aminotransferase, ASAT = aspartate aminotransferase.

Coupling of Enzyme Reactions and Mass Transfer in Immobilized Layers... 

Figure 14-26. Basic principles of the coupling of enzyme reactions in biosensors, a) Sequential coupling ...

From the analysis of the coupling of enzyme reaction and mass transfer, the following conclusions may be drawn for the design of biosensors. The substrate concentration at which deviations from the analytically usable linear measuring range occur depends on the extent of diffusion limitation. Under kinetic control, a linear dependence may only be expected for very low substrate concentrations. Under diffusion control, the decrease of substrate concentration in the enzyme layer caused by slow substrate diffusion results in an extended linear range. It has to be considered, however, that for two-substrate reactions deviations from linearity may also be produced by cosubstrate limitation. 

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] ... 

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. 

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. 

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 piezoelectrically-transduced biocatalytic biosensor that couples two enzyme reactions to detect glucose [492-62-6], C6H120 > (3) (13). In this biosensor a quartz radio crystal is functionalized with the enzyme glucose-6-phosphate dehydrogenase. As shown in Figure 3, a thin film of Prussian blue [14038 43-8], C18N18Fe7, is then coated onto the crystal. 

Interaction studies in isolated/cultured hepatocytes and liver slices have to be assessed critically since a couple of competing reactions occur e.g. uptake pathways or phase II metabolism of the NCE and/or marker substrate, which makes it extremely difficult to interpret the data mechanistically. Additionally, metabolic capacity of hepatocytes in culture change with time (e.g. decrease/increase of phase I and phase II enzyme activities and internalization of transporters). Usually, Michaelis-Menten kinetics does not apply directly. For reliable results, interactions studies have to be performed in hepatocytes from at least three different donors since pooled hepatocytes are not available yet. Similar problems are also discussed for interaction studies in liver slices (Ekins 1998). 

Under standard conditions, A cannot be spontaneously converted into B and C, because AG° is positive. However, the conversion of B into D under standard conditions is thermodynamically feasible. Because free-energy changes are additive, the conversion of A into C and D has a AG° of — 13kJmol ( —3kcalmoU ), which means that it can occur spontaneously under standard conditions. Thus, a thermodynamically unfavorable reaction can be driven by a thermodynamically favorable reaction to which it is coupled. In this example, the reactions are coupled by the shared chemical intermediate B. Thus, metabolic pathways are formed by the coupling of enzyme-catalyzed reactions such that the overall free energy of the pathway is negative. 

The theory of the coupling of enzyme-catalyzed reactions with transport processes has been investigated for the following limiting cases (Carr and Bowers, 1980) ... 

Fig. 89. Coupling of enzymatic reactions with electrochemical detection in amperometric enzyme electrodes for cholesterol. (Redrawn from Wollenberger et al., 1983).

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