NAD+ as coenzyme

Both the enzymes were prepared by a special technique from the insoluble portion of guinea pig liver mitochondria, and they are quite specific with respect to the requirement of pyridine nucleotide (H9, Hll). However, dehydrogenases catalyzing reaction (25) with NAD as coenzyme have been reported (Mil, S13, T3), thus confirming the importance of the source of the enzyme and the purification procedure employed. 

Because of the enzyme specificity in the hydration step, the new carbonyl group is introduced 3 to the original thioester carbonyl. The sequence includes two dehydrogenation reactions, and involves both FAD and NAD+ as coenzymes. The reduced forms of these coenzymes can be reoxidized by means of... 

Other enzymes may also be similarly inactivated by such cyclopropanone adducts generated in situ by catalytic unravelling of some latent precursors. NAD+ as coenzyme favours the electrophilic attack of AGP 17 on the enzymic thiol group, and considerably increases the rate of inhibition [17]. ALDH in brain was also inhibited in rats pretreated with coprine, and aldehyde reductase was slightly inhibited by AGP 17, in vitro [22]. 

The oxidations are performed in aqueous buffer solution at pH 9.0 with less than stoichiometric amounts of nicotineamide adenine dinucleotide (NAD+) as coenzyme, which is expensive and therefore must be recycled. This can be done in such oxidations in several ways. The simplest is to simultaneously oxidize the reduced nicotineamide adenine dinucleotide formed with a stoichiometric amount (based on the diol) of inexpensive flavin adenine mononucleotide (FMN)167. In this manner gram quantities of the corresponding lactone may be readily obtained scaling up the reaction, however, seems to be a problem167. 

In discussion of nomenclature of malic acid decomposing enzymes, mention should be made of malate-lactate transhydrogenase. This enzyme, isolated from Micrococcus lactiyticus (VielloneUa alcalescens), a bacterium found in vertibrates, can catalyze reversibly the conversion of L-malic and pyruvic acids to L-lactic and oxaloacetic acids with NAD as coenzyme (36). 

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. 

B. Riebel, W. Hummel, and A. Bommarius, Dehydrogenase mutants capable of using NAD as coenzyme and their preparation and use for chiral hydroxy compound preparation, Eur. Pat. Appl. EP 1176203 Al, 2002. 

The biochemical characterization of several alcohol dehydrogenases and their exploitation potential demonstrate that these enzymes are most important tools for biochemists. Amino acid sequences of several ADFls are available so far, and alignment studies allow to establish ADH families and to consider their probable evolutionary relationships. For preparative applications, however, particular properties of an enzyme are essential prerequisites, such as enzyme stability and availability, its substrate specificity, or reaction selectivity. Enzymes with NAD as coenzyme are clearly preferred to NADP-dependent ones in practice, because NAD has a significantly higher stability [186-188], a lower price and, is in general, easier to regenerate. 

Detailed initial rate studies at pH 7.0 have also been reported with ethanol and the acetylpyridine (APAD) and hypoxanthine (NHD) analogs of NAD as coenzyme (69,70). As would be expected for an ordered mechanism, all the kinetic coefficients are changed. With APAD, the maximum rate relations are, however, again consistent with a Theorell-Chance mechanism, but the maximum rate is significantly smaller than both the rate of dissociation of E-APADH measured directly by the stopped-flow method and the rate of hydride transfer measured from burst experiments. A rate-limiting isomerization of the E-APADH complex has been suggested (71), and is discussed in Section IV. 

Alcohol dehydrogenases are zinc enzymes that use NAD as coenzyme according to the reaction ... 

C, Estimated from Matthews and Widholm (1979a) inhibition was measured with 10 mM threonine and NAD as coenzyme. 

The third reaction of this cycle is the oxidation of the hydroxyl group at the /3-position to produce a /3-ketoacyl-CoA derivative. This second oxidation reaction is catalyzed by L-hydroxyacyl-CoA dehydrogenase, an enzyme that requires NAD as a coenzyme. NADH produced in this reaction represents metabolic energy. Each NADH produced in mitochondria by this reaction drives the synthesis of 2.5 molecules of ATP in the electron transport pathway. L-Hydroxyacyl-... 

Zincke salts have played an important role in the synthesis of NAD /NADH coenzyme analogs since a 1937 report on the Zincke synthesis of dihydropyridine 7 for use in a redox titration study.The widely utilized nicotinamide-derived Zincke salt 8, first synthesized by Lettre was also used by Shifrin in 1965 for the preparation and study of NAD /NADH analogs. In 1972, Secrist reported using 8 for synthesis of simplified NAD analogs such as 10 for use in spectroscopic studies (Scheme 8.4.4). Subsequent utilization of 8 is discussed later in this article. 

Step 3 of Figure 29.3 Alcohol Oxidation The /3-hydroxyacyl CoA from step 2 is oxidized to a /3-ketoacyl CoA in a reaction catalyzed by one of a family of L-3-hydroxyacyl-CoA dehydrogenases, which differ in substrate specificity according to the chain length of the acyl group. As in the oxidation of sn-glycerol 3-phosphate to dihydroxyacetone phosphate mentioned at the end of Section 29.2, this alcohol oxidation requires NAD+ as a coenzyme and yields reduced NADH/H+ as by-product. Deprotonation of the hydroxyl group is carried out by a histidine residue at the active site. 

Although fatty acids are both oxidized to acetyl-CoA and synthesized from acetyl-CoA, fatty acid oxidation is not the simple reverse of fatty acid biosynthesis but an entirely different process taking place in a separate compartment of the cell. The separation of fatty acid oxidation in mitochondria from biosynthesis in the cytosol allows each process to be individually controlled and integrated with tissue requirements. Each step in fatty acid oxidation involves acyl-CoA derivatives catalyzed by separate enzymes, utihzes NAD and FAD as coenzymes, and generates ATP. It is an aerobic process, requiring the presence of oxygen. 

Different preparative procedures have been shown to yield protein fractions which are able to catalyze different types of reactions with respect to their requirement of either NAD or NADP as coenzymes [cf. Eqs. (19), (20), and (21)]. In sera of mice poisoned by carbon tetrachloride we found polyol dehydrogenases catalyzing the oxidation of the following polyols (a) with NAD sorbitol, ribitol, mannitol (b) with NADP sorbitol, ribitol. Erythritol and mt/o-inositol were not attacked at all. Figures 8 and 9 show the results of these determinations performed at pH 9.6. In the NAD system sorbitol and ribitol are oxidized at exactly the same rate, while in the NADP system ribitol does not reach the rate of sorbitol. The ratio NAD NADP for sorbitol is calculated to be 4.20 and for ribitol 5.50. Mannitol is oxidized at 23% of the rate of sorbitol. 

Glutamate dehydrogenase, the enzyme responsible for the liberation of ammonia from amino acids, occurs in two forms one (cytosolic) is nicotinamide adenine dinucleotide (NAD+) dependent whilst the other (mitochondrial) requires NADP+ as coenzyme. 

Although the structures for molecules having niacin activity are simple, the forms in which they act in human biochemistry are not so simple. Nicotinic acid and nicotinamide are precursors for three complex coenzymes in multiple oxida-tion/reduction (redox) reactions nicotinamide mononucleotide, NMN nicotinamide adenine dinucleotide, NAD+ and nicotinamide adenine dinucleotide phosphate, NADP. I shall use NAD+ as representative of the class. NADH is the corresponding reduced form. ... 

In this reaction, pyruvic acid is oxidized to carbon dioxide with formation of acetyl-SCoA and NAD+ is reduced to NADH. As noted in chapter 15, this reaction requires the participation of thiamine pyrophosphate as coenzyme. Here too the NADH formed is converted back to NAD+ by the electron transport chain. As noted above, the acetyl-SCoA is consumed by the citric acid cycle and CoASH is regenerated. 

In this way, many biochemical systems can be linked together via a small number of pairs of compounds, with the fnnctions of X and Y, i.e. snch metabolic pairs play a major role in biochemistry. These compounds are known as coenzymes. Well-known examples include ADP/ATP, NAD+/NADH and NADF/NADPH. Such biochemical systems are discussed in Chapter 3. 

This sequence of reactions, namely oxidation of CH2-CH2 to CH=CH, then hydration to CH2-CHOH, followed by oxidation to CH2-CO, is a sequence we shall meet again in the -oxidation of fatty acids (see Section 15.4.1). The first oxidation utilizes FAD as coenzyme, the second NAD+. In both cases, participation of the oxidative phosphorylation system allows regeneration of the oxidized coenzyme and the subsequent generation of energy in the form of ATP. 

The pyridine nucleotides NAD"" and NADP" (1) are widely distributed as coenzymes of dehydrogenases. They transport hydride ions (2e and 1 see p. 32) and always act in soluble form. NAD" transfers reducing equivalents from catabolic pathways to the respiratory chain and thus contributes to energy... 

The five different coenzymes involved are associated with the enzyme components in different ways. Thiamine diphosphate is non-covalently bound to El, whereas lipoamide is covalently bound to a lysine residue of E2 and FAD is bound as a prosthetic group to E3. NAD" and coenzyme A, being soluble coenzymes, are only temporarily associated with the complex. 

This zinc metalloenzyme [EC 1.1.1.1 and EC 1.1.1.2] catalyzes the reversible oxidation of a broad spectrum of alcohol substrates and reduction of aldehyde substrates, usually with NAD+ as a coenzyme. The yeast and horse liver enzymes are probably the most extensively characterized oxidoreductases with respect to the reaction mechanism. Only one of two zinc ions is catalytically important, and the general mechanistic properties of the yeast and liver enzymes are similar, but not identical. Alcohol dehydrogenase can be regarded as a model enzyme system for the exploration of hydrogen kinetic isotope effects. 

Initial rate patterns for Escherichia coli NAD+-dependent coenzyme A-linked aldehyde dehydrogenase (Reaction NAD+ + CoA-SFI + acetaldehyde = NADFI + acetyl-S-CoA + FI+). The results of each of three experiments are shown as a single double-reciprocal plot, and the nonvaried substrate concentrations for each curve are indicated above the data points. 

Niacin is present in foods mainly as coenzyme NAD and NADP, which are hydrolyzed in the intestine, and it is adsorbed as nicotinamide or nicotinic acid. The free forms, nicotinamide and nicotinic acid, only allowed to be added in fortified foods [403], occur naturally in limited amounts. Instead, niacin occurs as nicotynil ester bonded to polysaccharides, peptides, and glycopeptides. In general, niacin is widespread in foodstuffs (cereals, seeds, meat, and fish). High concentrations are present in roasted coffee beans as a primarily product of the roasting process [417]. 

J.L. Panza, A.J. Russell, E.J. Beckman, Synthesis of fluorinated NAD as a soluble coenzyme for enzymatic chemistry in fluorous solvents and carbon dioxide. Tetrahedron 58 (2002) 4091-4104. 

Correlations with Hantzsch esters) (70) (77BSB267). This latter type of compound is of interest as an effective model for the NAD/NADH coenzyme system, and this work provides detailed analysis of H and 13C NMR of such compounds. 

The half-wave potential for the electrochemical oxidation of NADH to NAD is ca. -bO.6 V vj. SCE at pH 7. The formal potential for the NADH/NAD couple, however, is only —0.56 V. The overpotential therefore is about 1.2 V. As NAD acts as coenzyme in many enzyme-catalyzed oxidations of practical importance, it would be of interest to regenerate NAD electrochemically. For this purpose it is necessary to find a mediator system which is able to lower the overpotential. Mediator systems accepting two electrons or a hydride atom are most effective. Therefore, dopaquinone electro-generated from dopamine 2" and quinone diimines derived from diaminobenzenes applied successfully. 

TABLE 13-8 Stereospecificity of Dehydrogenases That Employ NAD+ or NADP+ as Coenzymes... 

The combined dehydrogenation and decarboxylation of pyruvate to the acetyl group of acetyl-CoA (Fig. 16-2) requires the sequential action of three different enzymes and five different coenzymes or prosthetic groups—thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), coenzyme A (CoA, sometimes denoted CoA-SH, to emphasize the role of the —SH group), nicotinamide adenine dinucleotide (NAD), and lipoate. Four different vitamins required in human nutrition are vital components of this system thiamine (in TPP), riboflavin (in FAD), niacin (in NAD), and pantothenate (in CoA). We have already described the roles of FAD and NAD as electron carriers (Chapter 13), and we have encountered TPP as the coenzyme of pyruvate decarboxylase (see Fig. 14-13). 

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