Alcohol-dehydrogenase-nicotinamide adenine dinucleotide

J. Wang, E. Gonzalez-Romero and M. Ozsoz, Renewable alcohol biosensors based on alcohol-dehydrogenase/nicotinamide-adenine-dinucleotide graphite epoxy electrodes, Electroanalysis, 4 (1992) 539-544. 

The substrate models concerned were fitted into the model of the active site of alcohol dehydrogenase-nicotinamide adenine dinucleotide (ADH-NAD) with VDW contacts, etc. not considered explicitly. 

Jagodzinski, P. W., Petlcolas, W. L. (1981) Resonance Enhanced Raman Identification of the Zinc-Oxygen Bond in a Horse Liver Alcohol Dehydrogenase-Nicotinamide Adenine Dinucleotide-Aldehyde Transient Chemical Intermediate, J. Am. Chem. Soc. 103, 234-236. 

Jagodzinski PW, Peticolas WL (1981) Resonance enhanced Raman identification of the zinc-oxygen bond in a horse liver alcohol dehydrogenase-nicotinamide adenine dinucleotide-aldehyde transient chemical intermediate. J Am Chem Soc 103 234-236 Jakovac IJ, Goodbrand HB, Lok KP, Jones JB (1982) Enzymes in organic synthesis. 24. Preparations of enantiomerically pure chiral lactones via stereospecific horse liver alcohol dehydrogenase... 

The difference between primary and secondary kinetic effects can be elucidated by using the oxidation of benzyl alcohol by nicotinamide adenine dinucleotide (NAD+) as an example (Scheme 10.1 (A)). This reaction is catalyzed by alcohol dehydrogenase (ADH), and has been extensively studied [10, 21-27]. In this reaction, the hydrogen at position is transferred from benzyl alcohol to NAD+, forming benzaldehyde and reduced nicotinamide (NADH), making the primary position. Conversely, is retained upon reaction, making this the secondary position. 

Cu-+ Peroxidase Cytochrome oxidase Nicotinamide adenine dinucleotide (NAD) Hydride ion (H ) Alcohol dehydrogenase... 

The leading substrate (A) is nicotinamide adenine dinucleotide (NAD ), and NAD and NADH (product Q) compete for a common site on E. A specific example is offered by alcohol dehydrogenase (ADH) ... 

Ethanol Electrodes The reliable sensing of ethanol is of great significance in various disciplines. The enzymatic reaction of ethanol with the cofactor nicotinamide-adenine dinucleotide (NAD+), in the presence of alcohol dehydrogenase (ADH)... 

Ethanol is oxidized by alcohol dehydrogenase (in the presence of nicotinamide adenine dinucleotide [NAD]) or the microsomal ethanol oxidizing system (MEOS) (in the presence of reduced nicotinamide adenine dinucleotide phosphate [NADPH]). Acetaldehyde, the first product in ethanol oxidation, is metabolized to acetic acid by aldehyde dehydrogenase in the presence of NAD. Acetic acid is broken down through the citric acid cycle to carbon dioxide (CO2) and water (H2O). Impairment of the metabolism of acetaldehyde to acetic acid is the major mechanism of action of disulfiram for the treatment of alcoholism. 

Zinc-containing alcohol dehydrogenases take up two electrons and a proton from alcohols in the form of a hydride. The hydride acceptor is usually NAD(P) (the oxidized form of nicotinamide adenine dinucleotide (NADH) or its phosphorylated derivative, NADPH). Several liver alcohol dehydrogenases have been structurally characterized, and Pig. 17.8 shows the environment around the catalytic Zn center and the bound NADH cofactor. 

Nicotinamide Adenine Dinucleotide (NAD+) Alcohol dehydrogenase, Lactate oxidase... 

Nicotinamide Adenine Dinucleotide phosphate (NADP+) Glutamate dehydrogenase, Alcohol dehydrogenase, Aryl-aldehyde dehydrogenase... 

It is possible to use isolated, partially purified enzymes (dehydrogenases) for the reduction of ketones to optically active secondary alcohols. However, a different set of complications arises. The new C H bond is formed by delivery of the hydrogen atom from an enzyme cofactor, nicotinamide adenine dinucleotide (phosphate) NAD(P) in its reduced form. The cofactor is too expensive to be used in a stoichiometric quantity and must be recycled in situ. Recycling methods are relatively simple, using a sacrificial alcohol, or a second enzyme (formate dehydrogenase is popular) but the real and apparent complexity of the ensuing process (Scheme 8)[331 provides too much of a disincentive to investigation by non-experts. 

A. Gafni and L. Brand, Fluorescence decay studies of reduced nicotinamide adenine dinucleotide in solution and bound to liver alcohol dehydrogenase, Biochemistry 15, 3165-3171 (1976). 

Figure 6.1 Pathways involved in glucose oxidation by plant cells (a) glycolysis, (b) Krebs cycle, (c) mitochondrial cytochrome chain. Under anoxic conditions. Reactions 1, 2 and 3 of glycolysis are catalysed by lactate dehydrogenase, pyruvate decarboxylase and alcohol dehydrogenase, respectively. ATP and ADP, adenosine tri- and diphosphate NAD and NADHa, oxidized and reduced forms of nicotinamide adenine dinucleotide PGA, phosphoglyceraldehyde PEP, phosphoenolpyruvate Acetyl-CoA, acetyl coenzyme A FP, flavoprotein cyt, cytochrome e, electron. (Modified from Fitter and Hay, 2002). Reprinted with permission from Elsevier...

NADH (reduced nicotinamide adenine dinucleotide) is utilized in biological reductions to deliver hydride to an aldehyde or ketone carbonyl group (see Box 7.6). A proton from water is used to complete the process, and the product is thus an alcohol. The reaction is catalysed by an enzyme called a dehydrogenase. The reverse reaction may also be catalysed by the enzyme, namely the oxidation of an alcohol to an aldehyde or ketone. It is this reverse reaction that provides the dehydrogenase nomenclature. 

NAD is one of Nature s most important oxidizing agents it can be considered as a biological equivalent of the chromium(VI) ion. NAD is shorthand for nicotinamide adenine dinucleotide it is a co-enzyme, which together with an enzyme is essential for several life-sustaining processes (Box 2.2). On reduction it forms the corresponding 1,4-dihydropyridine, NADH, The oxidation of ethanol to acetaldehyde (ethanal) is effected by the enzyme alcohol dehydrogenase and mediated by NAD (Scheme 2.31). 

Although zinc itself is not redox-active, some class I enzymes containing zinc in their active sites are known. The most prominent are probably alcohol dehydrogenase and copper-zinc superoxide dismutase (Cu,Zn-SOD). AU have in common that the redox-active agent is another transition-metal ion (copper in Cu,Zn-SOD) or a cofactor such as nicotinamide adenine dinucleotide (NAD+/NADH). The Zn(II) ion affects the redox reaction only in an indirect manner, but is nevCTtheless essential and cannot be regarded simply as a structural factor. 

Alcohol dehydrogenases (ADH EC 1.1.1.1), for which several X-ray structures are available ", catalyze the biological oxidation of primary and secondary alcohols via the formal transfer of a hydride anion to the oxidized form of nicotinamide adenine dinucleotide (NAD ), coupled with the release of a proton. Liver alcohol dehydrogenase (LADH) consists of two similar subunits, each of which contains two zinc sites, but only one site within each subunit is catalytically active. The catalytic zinc is coordinated in a distorted tetrahedral manner to a histidine residue, two cysteine residues and a water molecule. The remaining zinc is coordinated tetrahedrally to four cysteine residues and plays only a structural role . 

Metabolism of ethanol by alcohol dehydrogenase and the microsomal ethanol-oxidizing system (MEOS). Alcohol dehydrogenase and aldehyde dehydrogenase are inhibited by fomepizole and disulfiram, respectively. NAD +, nicotinamide adenine dinucleotide NADPH, nicotinamide adenine dinucleotide phosphate. 

RGURE 7 An oxidation-reduction reaction. Shown here is the oxidation of lactate to pyruvate. In this dehydrogenation, two electrons and two hydrogen ions (the equivalent of two hydrogen atoms) are removed from C-2 of lactate, an alcohol, to form pyruvate, a ketone. In cells the reaction is catalyzed by lactate dehydrogenase and the electrons are transferred to a cofactor called nicotinamide adenine dinucleotide. This reaction is fully reversible pyruvate can be reduced by electrons from the cofactor. In Chapter 13 we discuss the factors that determine the direction of a reaction. 

Ethanol also may be determined using alcohol dehydrogenase and measuring the change in nicotinamide adenine dinucleotide (NAD+) to the reduced form (NADH) at 340 nm. 

Several of the B vitamins function as coenzymes or as precursors of coenzymes some of these have been mentioned previously. Nicotinamide adenine dinucleotide (NAD) which, in conjunction with the enzyme alcohol dehydrogenase, oxidizes ethanol to ethanal (Section 15-6C), also is the oxidant in the citric acid cycle (Section 20-10B). The precursor to NAD is the B vitamin, niacin or nicotinic acid (Section 23-2). Riboflavin (vitamin B2) is a precursor of flavin adenine nucleotide FAD, a coenzyme in redox processes rather like NAD (Section 15-6C). Another example of a coenzyme is pyri-doxal (vitamin B6), mentioned in connection with the deamination and decarboxylation of amino acids (Section 25-5C). Yet another is coenzyme A (CoASH), which is essential for metabolism and biosynthesis (Sections 18-8F, 20-10B, and 30-5A). 

The alcohol dehydrogenases are zinc metalloenzymes which can oxidize a wide variety of alcohols to their corresponding aldehydes or ketones using nicotinamide adenine dinucleotide (NAD+) as coenzyme. These reactions are readily reversible so that carbonyl compounds may be reduced by NADH. 

We shall start the discussion with a classical experiment related to the stereochemistry of oxidation of ethanol and reduction of acetaldehyde mediated by the enzyme yeast alcohol dehydrogenase in the presence of the oxidized (NAD+) and reduced (NADH) forms, respectively, of the coenzyme nicotinamide adenine dinucleotide (Fig. 54). The stereochemically interesting feature of this reaction stems from the fact that the methylene hydrogens in CH3CH2OH and the faces of the carbonyl in CH3CH = 0 are enantiotopic. The question thus arises which of the CH2-hydrogens... 

Ethylene glycol is used as a freezing-point depressant in automotive antifreeze. It is highly toxic because the enzyme alcohol dehydrogenase and the coenzyme nicotinamide adenine dinucleotide (NAD) oxidize ethylene glycol to much more liver-toxic compounds like glyoxal, hydroxyacetaldehyde, glyoxylic... 

Horse liver alcohol dehydrogenase is a well-documented enzyme capable of operating with high stereoselectivity on a broad structural range of alcohol and carbonyl substrates. The present reaction proceeds via the pathway shown below, where NAD and NADH represent the oxidized and reduced forms, respectively, of the nicotinamide adenine dinucleotide coenzyme. 

Alcohol dehydrogenase catalyzes an oxidation the removal of two hydrogen atoms from the alcohol molecule. The oxidizing agent is nicotinamide adenine dinucleotide (NAD). NAD exists in two forms the oxidized form, called NAD+, and the reduced form, called NADH. The following equation shows that ethanol is oxidized to acetaldehyde, and NAD+ is reduced to NADH. 

Conditions for cytosolic incubations depend on the aim of the assay e.g. to cover specific enzymatic activity present in the cytosol. For this purpose it is essential to fortify the incubation medium with the specific cofactor for the reaction-if needed (Ekins 1999). (J> -Nicotinamide adenine dinucleotide (NAD) is needed for alcohol and aldehyde dehydrogenases, flavin adenine dinucleotide (FAD) for polyamine oxidase, P-nicotinamide adenine dinucleotide phosphate (NADPH) for Dihydropyrimidine dehydrogenase. Phase II reactions depend on PAPS (sulfotransferases,... 

Sloan, D.L., and Mildvan, A.S. (1974) Magnetic resonance studies of the geometry of bound nicotinamide adenine dinucleotide and isobutyramide on spin labeled alcohol dehydrogenase, Biochemistry 13, 1711-1718. 

The vast majority of alcohol dehydrogenases require nicotanimide cofactors, such as nicotinamide adenine dinucleotide (NADH) and its respective phosphate NADPH. The structure of NAD/NADP is shown in Fig. 3.39. Hydrogen and two electrons are transferred from the reduced nicotinamide to the carbonyl group to effect a reduction of the substrate (see Fig. 3.39). 

The 3-carbamidopyridinium ring is the chemically active portion of the enzymatic cofactors, NAD and NADP (nicotinamide adenine dinucleotide and its phosphate). A typical reaction involving NAD is the stereospecific (with respect to both cofactor and substrate) oxidation of ethanol to acetaldehyde catalyzed by the enzyme, alcohol dehydrogenase (Eq. 33). 

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