Enzyme coupled NADH

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. 

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. 

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... 

Fig. 7. Enzyme-coupled assay in which the hydrolase-catalyzed reaction releases acetic acid. The latter is converted by acetyl-CoA synthetase (ACS) into acetyl-CoA in the presence of (ATP) and coenzyme A (CoA). Citrate synthase (CS) catalyzes the reaction between acetyl-CoA and oxaloacetate to give citrate. The oxaloacetate required for this reaction is formed from L-malate and NAD in the presence of L-malate dehydrogenase (l-MDH). Initial rates of acetic acid formation can thus be determined by the increase in adsorption at 340 nm due to the increase in NADH concentration. Use of optically pure (Ry- or (5)-acetates allows the determination of the apparent enantioselectivity i app i81)-...

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... 

It should be mentioned that most natural aldolase enzymes can also be assayed using enzyme-coupled systems relaying the reaction to a redox process with NAD. The formation of NADH by active microbial colonies in expression libraries of mutant enzymes was detected colorimetrically in agar plates using phenazine methosulfate and nitroblue tetrazolium, which forms an insoluble precipitate. The assay was used by Williams et al. [14] and Woodhall et al. [15] for evolving sialic acid aldolases to accept non-natural aldehyde acceptors. 

Bioluminescence bioassays based on luminous bacteria and coupled enzyme system NADH-FMN-oxidoreductase-luciferase were adapted for monitoring the saline-water conditions of Lake Shira (Khakasia, Siberia). The differences in bioluminescence responses have been found to be related to the salt composition and the oxidation-reduction properties of water. Bioluminescent kinetics parameters, which are mostly sensitive to pollution under conditions of saline water, have been observed. Figure 1 shows the typical bioluminescence kinetics of the samples of water due to anthropogenic influence (beach) and control clear water (non-recreational area). 

FIGURE 17.11 Enzyme-catalyzed reduction of pyruvate to (S)-(+)-lactate. A preferred orientation of binding of pyruvate to the enzyme, coupled with a prescribed location of the reducing agent, the coenzyme NADH, leads to hydrogen transfer exclusively to a single face of the carbonyl group. 

The induction in B. megaterium by barbiturates, and the fact that the enzyme is unusual in having a single 119-kD protein coupling NADH reduction to oxygenation (Narhi and Fulco 1986). 

Figure 8.17 Enzyme coupled assay. Triose Phosphate Isomerase (TIM) assay system with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) colourimetric coupled assay for detection purposes. In this version of the TIM assay, dihydroxyacetone phosphate (DHAP) is converted enzymically to glyceraldehyde 3-phosphate (GAP) that is onward converted to glycerol phosphate (GP) by means of the coupled enzyme GAPDH enzyme that uses the reverse-colourimetric reductant NADH.

Xyhtol formation would decrease if recycling of NADH/NAD+ could take place in a single enzyme where NADH was oxidized at the XR site and reduced at the XDH site. Xylitol would then remain an enzyme-bound intermediate and the high microenviromnental concentration of NADH around the XR site would favor the utihzation of NADH. A series of XR and XDH fusion proteins were constructed [ 144]. The specific activities of XR and XDH depended on the order in which the two polypeptides were coupled in the hybrid protein, as well as on the length and composition of the connecting peptide. To obtain both XR and XDH activity, XDH had to be at the N-terminus and XR at the C-terminus of the fusion protein. Constructs with the opposite order lacked XR activity. The specific XDH activity increased threefold in the construct containing a linker consisting of 12 amino-acid residues, compared with a 7-residue hnker, while the XR activity remained constant. 

Another interesting example of a redox neutral cascade has been proposed for the multienzymatic synthesis of (JJ)-3-fluorolactic acid together with the resolution of racemic 3-fluoroalanine (Scheme 11.5b) [13]. Optically enriched (S)-3-fluoroalanine (88% ee) was recovered unreacted after the enantioselective oxidative deamination of the racemic substrate catalyzed by the L-alanine dehydrogenase (i-AlaDH) from Bacillus subtilis. This oxidative reaction, which is thermodynamically unfavorable, was driven by the coupled reduction reaction of the intermediate 3-fluoropyruvate catalyzed by rabbit muscle i-lactate dehydrogenase (L-LDH). Since both enzymes are NADH dependent, this coupled... 

In 2011, alcohol dehydrogenase (ADH) was used as a model enzyme coupled with poly (MG) for NADH reoxidation in the construction of a three-dimensional BFC with ethanol as fuel [103]. In combination with an air-breathing/gas difhision cathode (using laccase as an oxygen reduction enzyme), a BFC was fabricated that was able to successfully exploit ethanol oxidation by an NAD -dependent ADH, immobilized by entrapment in a multiwaUed CNT (MWCNT)/chitosan matrix [106]. The feasibility and reproducibUity of the resulting BFC were demonstrated in 2008 with a series of standardized multilaboratory experiments [96]. 

This is a crucial point because (as we will see) proton transport is coupled with ATP synthesis. Oxidation of one FADHg in the electron transport chain results in synthesis of approximately two molecules of ATP, compared with the approximately three ATPs produced by the oxidation of one NADH. Other enzymes can also supply electrons to UQ, including mitochondrial 5w-glyc-erophosphate dehydrogenase, an inner membrane-bound shuttle enzyme, and the fatty acyl-CoA dehydrogenases, three soluble matrix enzymes involved in fatty acid oxidation (Figure 21.7 also see Chapter 24). The path of electrons from succinate to UQ is shown in Figure 21.8. 

This impressive reaction is catalyzed by stearoyl-CoA desaturase, a 53-kD enzyme containing a nonheme iron center. NADH and oxygen (Og) are required, as are two other proteins cytochrome 65 reductase (a 43-kD flavo-protein) and cytochrome 65 (16.7 kD). All three proteins are associated with the endoplasmic reticulum membrane. Cytochrome reductase transfers a pair of electrons from NADH through FAD to cytochrome (Figure 25.14). Oxidation of reduced cytochrome be, is coupled to reduction of nonheme Fe to Fe in the desaturase. The Fe accepts a pair of electrons (one at a time in a cycle) from cytochrome b and creates a cis double bond at the 9,10-posi-tion of the stearoyl-CoA substrate. Og is the terminal electron acceptor in this fatty acyl desaturation cycle. Note that two water molecules are made, which means that four electrons are transferred overall. Two of these come through the reaction sequence from NADH, and two come from the fatty acyl substrate that is being dehydrogenated. 

Energy-linked transhydrogenase, a protein in the inner mitochondrial membrane, couples the passage of protons down the electrochemical gradient from outside to inside the mitochondrion with the transfer of H from intramitochondrial NADH to NADPH for intramitochondrial enzymes such as glutamate dehydrogenase and hydroxylases involved in steroid synthesis. 

CK catalyzes the reversible phosphorylation of creatine in the presence of ATP and magnesium. When creatine phosphate is the substrate, the resulting creatine can be measured as the ninhydrin fluorescent compound, as in the continuous flow Auto Analyzer method. Kinetic methods based on coupled enzymatic reactions are also popular. Tanzer and Gilvarg (40) developed a kinetic method using the two exogenous enzymes pyruvate kinase and lactate dehydrogenase to measure the CK rate by following the oxidation of NADH. In this procedure the main reaction is run in a less favorable direction. 

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. 

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