Coupling enzyme

Figure 18.6 Energetics of the ORR at the heme/Cu site of CcO the enzyme couples oxidation of ferroc3ftochrome c (standard potential about —250 mV all potentials are listed with respect to a normal hydrogen electrode) to reduction of O2 (standard potential at pH 7 800 mV). Of the 550 mV difference, only 100 mV is dissipated to drive the reaction 220 mV is expanded to translocate four protons from the basic matrix compartment to the acidic IMS (inter-membrane space). In addition 200 mV is converted into transmembrane electrostatic potential as ferroc3ftochrome is oxidized in the IMS, but the charge-compensating protons are taken from the matrix. The potentials are approximate.

The flavin reductase has been purified by several researchers. This enzyme from R. erythropolis IGTS8 was partially purified by Ohshiro et al., and reported to have an optimum pH and temperature of 6.0 and 35°C, respectively [153], The DszD enzyme from IGTS8 was also purified [53] and reported to be of 25 kDa size however, no kinetic details related to DszD were reported. This enzyme couples with FMN with NADH to produce reduced flavin required for DszC and DszA catalyzed reactions. 

Kitagawa, T., and Aikawa, T. (1976) Enzyme coupled immunoassay of insulin using a novel coupling reagent. J. Biochem. (Tokyo) 79, 233-236. 

Enzyme coupled receptors Signal activates an enzyme activity of the receptor itself—tyrosine kinases, phospholipase C. 

For enzyme coupled receptors, activation of the receptor turns the receptor itself into an active enzyme. This activity may belong to the receptor itself, but sometimes activation of the receptor recruits and activates a separate enzyme through adaptor molecules (Fig. 9-3). A common mechanism of activation of these receptors involves dimerization. The signal molecule causes individual molecules of the receptor to associate with themselves in the membrane. Once dimerized, the receptors become activated and gain enzyme activity. 

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. 

Another type of sensor was based on the utilization of glucose dehydrogenase enzyme coupling with /ra(2,2 -bipyridylruthenium(II) complex [31]. This sensor can be used in the 10-2500-pmol/L concentration range. Several interferences occur, like NADH, oxalate, proline, and tripropylamine. However, gluconic acid and NAD+ do not interfere. 

A more successful strategy for developing sensitive and facile assays to monitor PLCBc activity involves converting the phosphorylated headgroup into a colorimetric agent via a series of enzyme coupled reactions. For example, phosphatidylcholine hydrolysis can be easily monitored in a rapid and sensitive manner by enzymatically converting the phosphorylcholine product into a red dye through the sequential action of alkaline phosphatase, choline oxidase, and peroxidase [33]. This assay, in which 10 nmol of phosphorylcholine can be readily detected, may be executed in a 96-well format and has been utilized in deuterium isotope and solvent viscosity studies [34] and to evaluate inhibitors of PLCBc [33] and site-directed mutants of PLCBc [35,36]. 

Enzymatic cofactor regeneration can be subdivided into two categories the enzyme-coupled approach, where two different enzymes are used (one for the production reaction, and one for the regeneration reaction) and the substrate-coupled approach, where one and the same enzyme is used for both production and regeneration (E = E2). The most convenient and commonly used enzymatic regeneration systems are summarized in Table 43.1. 

Therefore, for preparative applications of redox enzymes, effective and simple methods for the continuous recycling of the active cofactors have to be available. In addition, such systems must be stable over long time periods and the separation of the product must be simple to render technical processes economically feasible. Until now, this problem has generally been solved by the application of a second enzymatic reaction (enzyme-coupled regeneration, Fig. 2). 

Fig. 2, Principal of an enzyme-coupled cofactor regeneration system for an enzymatic reduction...

L-Pipecolic acid, a key component of many antibiotic and anticancer biomolecules, serves as an important chiral pharmaceutical intermediate. We have developed an enzyme-coupled system consisting of zl -piperidine-2-carboxylate reductase (Pip2C) from Pseudomonas putida, glucose dehydrogenase (GDH) from Bacillus subtilis, and L-lysine a-oxidase from Trichoderma viride, affording L-pipecolic acid from L-lysine in high yield with an excellent enantioselectivity (Figure 10.2). ... 

Figure 10.2 Enzyme-coupled system for synthesis of r-pipecoUc acid from L-lysine...

A second example of a membrane-bound arsenate reductase was isolated from Sulfurospirillum barnesii and was determined to be a aiPiyi-heterotrimic enzyme complex (Newman et al. 1998). The enzyme has a composite molecular mass of 100kDa, and a-, P-, and y-subunits have masses of 65, 31, and 22, respectively. This enzyme couples the reduction of As(V) to As(III) by oxidation of methyl viologen, with an apparent Kra of 0.2 mM. Preliminary compositional analysis suggests that iron-sulfur and molybdenum prosthetic groups are present. Associated with the membrane of S. barnesii is a h-type cytochrome, and the arsenate reductase is proposed to be linked to the electron-transport system of the plasma membrane. 

Bioprocesses incorporating more than one redox enzyme in an oxidative reaction system might involve, in the simplest case, two oxidizing enzymes coupled so that they act sequentially to effect two oxidation steps. A key issue in the development of such oxidative biocatalytic systems would be the determination of the values, for each enzyme involved, of the redox potentials. These can be determined by potentiometric titration using redox mediators (such as NADH) and techniques such as cyclic voltammetry or electrophoresis [44]. Knowledge of the redox potentials would facilitate the design and engineering of a process in which the two... 

In the second approach the reducing equivalents are suppHed by a nicotinamide cofactor (NADH or NADPH) and for commercial viability it is necessary to regenerate the cofactor using a sacrificial reductant ]12]. This can be achieved in two ways substrate coupled or enzyme coupled (Scheme 6.2). Substrate-coupled regeneration involves the use of a second alcohol (e.g. isopropanol) that can be accommodated by the KRED in the oxidative mode. A problem with this approach is that it affords an equilibrium mixture of the two alcohols and two ketones. In order to obtain a high yield of the desired alcohol product a large excess of the sacrificial alcohol needs to be added and/or the ketone product (acetone) removed... 

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