NADH-specific reduction

In an early report to a process using three oxidoreductases, namely hydrogenase (ECl.12.2.1), lipoamide dehydrogenase (EC 1.6.4.3) and 20(3-hydroxysteroid dehydrogenase (ECl.1.1.53), a reverse micelle system was used to facilitate stereo- and site-specific reduction of apolar ketosteroids, assisted by the in situ NADH-regenerating enzyme system [61]. 

In order to enhance affinity and selectivity for Brc-Abl, we modified the inhibitor methylating at positions I and II (Fig. 7.5d). The synthesis of the wrapping prototype recapitulates imatinib synthesis [38], as described in [39], To test whether the specificity and affinity for Brc-Abl improved, we conducted a spectrophotometric kinetic assay to measure the phosphorylation rate of peptide substrates in the presence of the kinase inhibitor at different concentrations. This assay couples production of adenosine diphosphate (ADP), the byproduct of downstream phosphorylation, with the concurrent detectable oxidation of reduced nicotinamide adenosine dinucleotide (NADH). The oxidation results upon transfer of phosphate from PEP (phospho-enolpyruvate) to ADP followed by the NADH-mediated reduction of PEP to lactate. Thus, phosphorylation activity is monitored by the decrease in 340 nm absorbance due to the oxidative conversion NADH->-NAD+ [34, 39]. 

Jolly et al, 1976 Shen et al., 1976) have succeeded in separating two enzymes, one of which shows specificity for NADH as reductant and a second present in small amounts, which is functional with both NADH and NADPH. 

Further structural diversification of FruA products has been investigated by enzymatic reduction to corresponding alditols using stereochemically complementary alditol dehydrogenases (Figure 5.40). Indeed, stereospecific (2S)- and (2R)-specific reduction of simple derivatives of o-fructose could be achieved by NADH-dependent catalysis of sorbitol dehydrogenase (EC 1.1.1.14) [209] or mannitol dehydrogenase (EC 1.1.1.67), respectively [26]. 

The bioluminescent determinations of ethanol, sorbitol, L-lactate and oxaloacetate have been performed with coupled enzymatic systems involving the specific suitable enzymes (Figure 5). The ethanol, sorbitol and lactate assays involved the enzymatic oxidation of these substrates with the concomitant reduction of NAD+ in NADH, which is in turn reoxidized by the bioluminescence bacterial system. Thus, the assay of these compounds could be performed in a one-step procedure, in the presence of NAD+ in excess. Conversely, the oxaloacetate measurement involved the simultaneous consumption of NADH by malate dehydrogenase and bacterial oxidoreductase and was therefore conducted in two steps. 

As a consequence of the previous considerations Kieber et al. [75] have developed an enzymic method to quantify formic acid in non-saline water samples at sub-micromolar concentrations. The method is based on the oxidation of formate by formate dehydrogenase with corresponding reduction of /3-nicotinamide adenine dinucleotide (j6-NAD+) to reduced -NAD+(/3-NADH) jS-NADH is quantified by reversed-phase high performance liquid chromatography with fluorimetric detection. An important feature of this method is that the enzymic reaction occurs directly in aqueous media, even seawater, and does not require sample pre-treatment other than simple filtration. The reaction proceeds at room temperature at a slightly alkaline pH (7.5-8.5), and is specific for formate with a detection limit of 0.5 im (SIN = 4) for a 200 xl injection. The precision of the method was 4.6% relative standard deviation (n = 6) for a 0.6 xM standard addition of formate to Sargasso seawater. Average re-... 

Using two types of specially synthesized rhodium-complexes (12a/12b), pyruvate is chemically hydrogenated to produce racemic lactate. Within the mixture, both a d- and L-specific lactate dehydrogenase (d-/l-LDH) are co-immobilized, which oxidize the lactate back to pyruvate while reducing NAD+ to NADH (Scheme 43.4). The reduced cofactor is then used by the producing enzyme (ADH from horse liver, HL-ADH), to reduce a ketone to an alcohol. Two examples have been examined. The first example is the reduction of cyclohexanone to cyclohexanol, which proceeded to 100% conversion after 8 days, resulting in total TONs (TTNs) of 1500 for the Rh-complexes 12 and 50 for NAD. The second example concerns the reduction of ( )-2-norbornanone to 72% endo-norbor-nanol (38% ee) and 28% exo-norbornanol (>99% ee), which was also completed in 8 days, and resulted in the same TTNs as for the first case. 

This was confirmed by Keilin and Hartree using antimycin A as an inhibitor. The antibiotic blocked the reduction of cytochrome cx by NADH or succinate but did not block the reduction of cytochrome b. This site-specific inhibition brought antimycin A into popular use by biochemists in the analysis of electron transfer and oxidative phosphorylation. 

Clostridium sticklandii also expresses a proline reductase that can reduc-tively cleave proline to 8-aminovalerate (Seto and Stadtman 1976). PR was first purified by Seto and Stadtman (1976) by following the decomposition of proUne in the presence of dithiothreitol or NADH. They found PR to have a denatured mass of approximately 30kDa (sodium dodecyl sulfate-polyacrylomide gel electrophoresis SDS-PAGE) and a native size of approximately 300 kDa. The addition of selenite to the growth medium of C sticklandii did increase the specific activity of PR in extracts by threefold however, no selenium was detected in the purified enzyme. It should be noted that this purified enzyme had lost the ability to couple reduction of proline to NADH and thus probably was missing one or more components of the complete enzyme complex. 

This process is commonly referred to as assimilatory nitrogen (nitrate or nitrite) reduction. The electrons for these reductions are supplied by half-cell oxidations involving NADPH/NADP" and NADH/NAD" (Table 7.11). All of these reactions and membrane transport processes are mediated by enzymes that are specific to the DIN species. Considerable variation exists among the phytoplankton species in their ability to produce the necessary enzymes. Since marine phytoplankton are often nitrogen limited, the quantity and type of DIN available in the water column can greatly influence overall phytoplankton abundance and species diversity. 

Surface fluorescence of NADH/NADPH can be recorded continuously with a DC fluorimeter and correlated with changes in experimental conditions. A mercury arc lamp (with a 340-375 nm filter in front) is used as a hght source for fluorescence excitation. The fluorescence response of reduced NADH/NADPH was measured at 450-510 nm. The DC fluorimeter and the Hg arc lamp are connected to the kidney by a trifurcated fiber optics light guide. NADH/NADPH fluorescence emission can be corrected for changes in tissue opacity by a 1 1 subtraction of reflectance changes at 340-375 nm from the fluorescence. To determine NADH/NADPH redox state of the total surface area of kidney cortex and to evaluate whether certain areas were insufficiently perfused, fluorescence photographs of the total surface area were taken. The study demonstrated that the surface fluorescence method is simple and provides specific information about the mitochondrial oxidation-reduction state. 

Two pyridine nucleotide-specific dehydrogenases are responsible for oxygen reduction in the cytosol a highly active NADH oxidase that reduces oxygen to water (Linstead and Bradley, 1988 Tanabe, 1979) and a minor NADPH oxidase that produces hydrogen peroxide (Linstead and Bradley 1988). 

The development by Chance of a dual wavelength spectrophotometer permitted easy observation of the state of oxidation or reduction of a given carrier within mitochondria.60 This technique, together with the study of specific inhibitors (some of which are indicated in Fig. 18-5 and Table 18-4), allowed some electron transport sequences to be assigned. For example, blockage with rotenone and amytal prevented reduction of the cytochrome system by NADH but allowed reduction by succinate and by other substrates having their own flavoprotein components in the chain. Artificial electron acceptors, some of which are shown in Table 18-5,... 

Use the plot of AiAQ vs. time to calculate AH/min over the linear portion of the curve. Convert the rate in absorbance terms to activity units. One enzyme unit is the amount of malate dehydrogenase that catalyzes the reduction of 1 micromole of oxaloacetate to L-malate in 1 minute under the described assay conditions. The reduction of 1 micromole of oxaloacetate leads to the oxidation of 1 micromole of NADH therefore, Equation E10.4 may be used to calculate the specific activity of malate dehydrogenase. 

Also, oxidation of an aldehyde to an acid is accomplished with NAD . There is a related reaction in photosynthesis (Section 20-9) that accomplishes the reduction of an acid to an aldehyde and is specific for NADPH, not NADH ... 

This scheme was supported and refined by examining the effects of specific inhibitors of individual steps in the electron-transport chain. If CO or CN was added in the presence of a reducing substrate and 02, all of the electron carriers became more reduced. This fits the idea that these inhibitors act at the end of the respiratory chain, preventing the transfer of electrons from cytochrome to 02. If amytal (a barbiturate) or rotenone (a plant toxin long used as a fish poison) was added instead, NAD+ and the flavin in NADH dehydrogenase were reduced, but the carriers downstream became oxidized. The antibiotic antimycin caused NAD+, flavins, and the b cytochromes to become more reduced, but cytochromes c, cx, a, and a3 all became more oxidized. The situation here is analogous to the construction of a dam across a stream When the gates are closed, the water level rises upstream from the dam, and falls downstream. The observation that antimycin did not inhibit reduction of UQ showed that the quinone fits into the chain upstream of cytochromes c, t i, a, and a3. 

Enzymes present in mammalian liver are capable of the catabolism of both uracil and thymine. The first reduces uracil and thymine to the corresponding 5,6-dihydro derivatives. This hepatic enzyme uses NADPH as the reductant, whereas a similar bacterial enzyme is specific for NADH. Similar enzymes are apparently present in yeast and plants. Hydropyrimidine hydrase then opens the reduced pyrimidine ring, and finally the carbamoyl group is hydrolyzed off from the product to yield /3-alanine or /3-aminoisobutyric acid, respectively, from uracil and thymine (see fig. 23.23). 

A different approach for utilization of the photoproducts in chemical routes involves the introduction of natural enzymes as catalysts in the photochemical system. In nature, dihydronicotinamide adenine dinucleotide (NADH) and dihydronicotinamide dinucleotide phosphate (NADPH) participate as reducing cofactors in a variety of enzymatic reduction processes. Thus, the development of photochemical NADH and NADPH regeneration cycles is anticipated to allow a variety of reduction processes by inclusion of substrate specific NAD(P)H dependent enzymes. 

Oxidation is by far the most important Phase I metabolic reaction. One of the main enzyme systems involved in the oxidation of xenobiotics appears to be the so called mixed function oxidases or monooxygenases, which are found mainly in the smooth endoplasmic reticulum of the liver but also occur, to a lesser extent, in other tissues. These enzymes tend to be nonspecific, catalysing the metabolism of a wide variety of compounds (Table 9.2). Two common mixed function oxidase systems are the cytochrome P-450 (CYP-450) and the flavin monoxygenase (FMO) systems (Appendix 12). The overall oxidations of these systems take place in a series of oxidative and reductive steps, each step being catalysed by a specific enzyme. Many of these steps require the presence of molecular oxygen and either NADH or NADPH as co-enzymes. 

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