NAD +-dependent enzymes

A NOVEL STRATEGY FOR THE DEVELOPMENT OF BIOSENSORS BASED ON NAD-DEPENDENT ENZYMES... 

A lot of analytical techniques have been proposed in recent decades and most of them are based on enzymes, called dehydrogenases, which are not sensitive to oxygen and need cofactors such as NAD". The key problems which seriously hamper a wide commercialization of biosensors and enzymatic kits based on NAD-dependent enzymes are necessity to add exogenous cofactor (NAD" ) into the samples to be analyzed to incorporate into the biologically active membrane of sensors covalently bounded NAD" to supply the analytical technique by NAD -regeneration systems. 

NAD+ and NADP+ are coenzymes of dehydrogenases. NADH and NADPH are intermediate carriers of both hydrogen and electrons. Most NAD-dependent enzymes are located in the mitochondria and deliver H2 to the respiratory chain whereas NADP-dependent enzymes take part in cytosolic syntheses (reductive biosyntheses). 

The oxidation of fatty acids within the Knoop-Lynen cycle occurs in the matrix. The Knoop-Lynen cycle includes four enzymes that act successively on acetyl-CoA. These are acyl-CoA dehydrogenase (FAD-dependent enzyme), enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase (NAD-dependent enzyme), and acetyl-CoA acyltrans-ferase. Each turn, or revolution, of the fatty acid spiral produces... 

Oppenheimer NJ, Handlon AL (1992) Mechanism of NAD-dependent enzymes. In Sigman DS (ed) The enzymes, Academic Press, San Diego, p 453... 

This approach was coupled to a system of three NAD+-dependent enzymes comprised of alcohol dehydrogenase (EC 1.1.1.1), aldehyde dehydrogenase (EC 1.2.1.3), and formate dehydrogenase (EC 1.2.1.2) to create an electrode theoretically capable of complete oxidation of methanol to carbon dioxide, as shown in Eigure 5. The anode was, in turn, coupled to a platinum-catalyzed oxygen cathode to produce a complete fuel cell operating at pH 7.5. With no externally applied convection, the cell produced power densities of 0.67 mW/cm at 0.49 V for periods of less than 1 min, before the onset of concentration polarization. 

In humans, 18 HDACs have been identified and classified according to their homology to yeast HDACs [6]. Class I, II and IV HDACs are zinc-dependent enzymes, whereas the third class (sirtuins) are NAD -dependent enzymes and are covered elsewhere in this book. Class I (H DACs 1, 2, 3, 8) are closely related to yeast Rpd3 class Ila (HDACs 4, 5, 7, 9) and class Ilb (HDACs 6, 10) are related to yeast Hdal and this latter subclass contains two catalytic sites. Finally, class IV H DACs contain just one member (HDAC 11). Whilst classes I and IV HDACs are mainly found in the nucleus of cells, class II H DACs are free to shuttle between the nucleus and the cytoplasm. The exact physiological role of each of the individual H DAC isoforms in cells is far from fully understood, yet it is known that these enzymes act on many other nonhistone substrates. They also often function as part of larger multiprotein complexes and are frequently associated with other HDAC isoforms and/or require the presence of several coregulators. 

NAD GLYCOHYDROLASE NAD -dependent dehydrogenases, ABSORPTION SPECTROSCOPY NAD -dependent enzymes,... 

Reactions catalyzed by 11 (3-hydroxysteroid and 17(3-hydroxysteroid dehydrogenases, (a) 11 (3-hydroxysteroid dehydrogenase type 1, an NADPH-dependent enzyme, catalyzes the conversion of the inactive steroid, cortisone, to cortisol, which is the biologically active glucocorticoid. 11 (3-hydroxysteroid dehydrogenase type 2, an NAD+-dependent enzyme, catalyzes the reverse direction, (b) 17(3-hydroxysteroid dehy-drogenase type 1, an NADPH-dependent enzyme, catalyzes the reduction of estrone to estradiol. Type 2, an NAD+-dependent enzyme, catalyzes the oxidation of estradiol to estrone. Type 3, an NADPH-dependent enzyme, catalyzes the reduction of androstene dione to testosterone. Type 4, an NAD+-dependent enzyme, catalyzes the oxidation of estradiol to estrone, and androstenediol to dehydroepiandrosterone. 

There are two different forms of isocitrate dehydrogenase in all cells, one requiring NAD+ as electron acceptor and the other requiring NADP+. The overall reactions are otherwise identical. In eukaryotic cells, the NAD-dependent enzyme occurs in the mitochondrial matrix and serves in the citric acid cycle. The main function of the NADP-dependent enzyme, found in both the... 

The most confusing aspect of the pathway proposed by Ochoa and his group now rests with the NAD requirement. In proceeding from L-malic acid to L-lactic acid, there is no net change in oxidation state. Yet in whole cells or cell-free extracts, the malo-lactic fermentation will not proceed in the absence of NAD. Therefore, by the proposed mechanism, one is unable to demonstrate the appearance of reduced cofactor, and the NAD specificity cannot be explained as a redox requirement. However, in the time since this mechanism was proposed, an NAD dependent enzyme (glyceraldehyde-3-phosphate dehydrogenase) has been described which requires NAD in a non-redox capacity (29), and it is possible that the same is true for the enzyme causing the malic acid-lactic acid transformation. 

If there are three or more -SH groups in a chain some incorrect pairing may, and often does, occur. Tire protein disulfide isomerases break these bonds and allow new ones to form.92 The active sites of these isomerases contain pairs of -SH groups which can be oxidized to internal -S-S- bridges by NAD+-dependent enzymes. These enzymes and their relatives thioredoxin and glutaredoxin are discussed further in Box 15-C. Glutathione and oxidation-reduction buffering are considered in Box 11-B. 

NAD+ affinity tag. By offering an NAD+-laden column, or more often a 2-ADP-Sepharose column, to an NAD+-dependent enzyme such as a dehydrogenase, such enzymes are bound while all others are passing through the column. Subsequent elution with 0.5 mM NAD+ after a washing step to remove unspecifically bound protein results in a relatively pure fraction of protein which should contain only NAD+-dependent proteins. 

This NAD-dependent enzyme was purified up to a specific activity of 1060 U/mg (diacetyl as substrate). The enzyme is stable at 57°C for 10 min, the temperature optimum is at 70°C. Besides diacetyl several other diketones were reduced. 

Nicotinamide adenine dinucleotide is a coenzyme which is only loosely bound to the active site of the enzymes with which it interacts and is free therefore, to dissociate from the enzyme during the catalytic cycle. The role of the dehydrogenase enzyme is to bring together the substrate and the NAD+ in the correct orientation for the two to react. These NAD+-dependent enzymes are known as dehydrogenases. They work in conjunction with NAD+ to oxidise substrates by the transfer of 1H+ and 2e from the substrate to the 4-position of the nicotinamide ring of the NAD+ (see Fig. 2.1). The overall reaction is the equivalent of a hydride transfer and is commonly referred to as such. NAD+-dependent enzymes are primarily involved in respiration (NAD+ occurs in significant amounts in mitochondria), whereas, NADP+-dependent coenzymes are primarily involved in the transfer of electrons from intermediates in catabolism. 

A general method has been developed for utilization of cofactor-requiring enzymes in organic media [139]. ADH from horse liver as well as NADH were attached onto the surface of glass beads and afterwards suspended in a water-immiscible organic solvent containing the substrate. This method can be applied to other NAD+-dependent enzymes as well. Both NADH and NAD+ are efficiently regenerated with ADH-catalyzed oxidation of ethanol and reduction of isobutyr-aldehyde, respectively (Fig. 31). 

In primary structure, the NADP-dependent N. crassa enzyme shows clear but limited homology with the vertebrate glutamate dehydrogenases in the N-terminal two-thirds of the chain [98]. Interestingly, the NAD-dependent enzyme of N. crassa has a much larger subunit, with an apparently dissimilar sequence [99],... 

Consider the NAD -dependent enzyme dehydrogenase, which catalyzes the oxidation of a substrate, RH ... 

Inositol can also be synthesized endogenously inositol 1-phosphate is formed by isomerization of glucose 6-phosphate, catalyzed by an NAD-dependent enzyme, although overall there is no change in redox state. Phos-phatidylinositol is formed by a reaction between CDP-inositol and diacylglyc-erol. Most inositol is cataboUzed by oxidation to glucuronic acid. 

Cytochrome P450 Monooxygenases, Chemistry of Enzyme Catalysis, Roles of Structural Dynamics in Enzyme Catalysis, Chemical Strategies for NAD+ Dependent Enzymes, Chemistry of Oxygen-Activating Enzymes, Chemistry of Transient State Enzyme Kinetics Flavoenzymes, Chemistry of... 

In the second step, acetaldehyde is reduced to ethanol by alcohol dehydrogenase, an NAD-dependent enzyme. 

Ethanol oxidation begins with conversion to acetaldehyde by alcohol dehydrogenase (M.W. 85,000), a zinc-containing, NAD+-dependent enzyme that is a relatively nonspecific cytoplasmic enzyme with a of about 1 mM/L ... 

The alignment and electrical contacting of NAD -dependent enzymes on electrodes was also accomplished by the generation of the NAD enzytne complex and its crosslinking on a conductive, redox-active, polymer that electrically contacts the cofactor-enzyme assembly with the electrode, Fig. 3-23. 

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