Nicotinamide active sites

Oxidation of P-nicotinamide adenine dinucleotide (NADH) to NAD+ has attracted much interest from the viewpoint of its role in biosensors reactions. It has been reported that several quinone derivatives and polymerized redox dyes, such as phenoxazine and phenothiazine derivatives, possess catalytic activities for the oxidation of NADH and have been used for dehydrogenase biosensors development [1, 2]. Flavins (contain in chemical structure isoalloxazine ring) are the prosthetic groups responsible for NAD+/NADH conversion in the active sites of some dehydrogenase enzymes. Upon the electropolymerization of flavin derivatives, the effective catalysts of NAD+/NADH regeneration, which mimic the NADH-dehydrogenase activity, would be synthesized [3]. 

What accounts for this stereospecificity It arises from the fact that the enzymes (and especially the active sites of enzymes) are inherently asymmetric structures. The nicotinamide coenzyme (and the substrate) fit the active site in only one way. Malate... 

Figure 17-5. Oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase complex. Lipoic acid is joined by an amide link to a lysine residue of the transacetylase component of the enzyme complex. It forms a long flexible arm, allowing the lipoic acid prosthetic group to rotate sequentially between the active sites of each of the enzymes of the complex. (NAD nicotinamide adenine dinucleotide FAD, flavin adenine dinucleotide TDP, thiamin diphosphate.)...

Figure 3.1 Amino add side-chain groups involved in binding NAD at the active site of an enzyme. The enzyme is glyceraldehyde dehydrogenase. More than 20 amino acids, the position of which in the primary structure is indicated by the number, counting from the N-terminal amino acid, are involved in the binding. This emphasises the complexity of the binding that is responsible for the specificity of the enzyme for NAD (depicted in bold). The molecular structure of nicotinamide adenine dinucleotide (NAD ) provided in Appendix 3.3.

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. 

The nicotinamide moiety that defines the active site of the enzyme is seen in the center. 

Inhibition studies involving ALR2 have indicated noncompetitive inhibition for virtually all compounds examined to date when the forward (reduction) reaction is monitored. This mode of inhibition is often interpreted as meaning that the inhibitor binds to a site on the enzyme that is independent of the catalytic site. Kinetic and competition studies have both led to this conclusion in the case of ALR2 [24,25]. The crystal structure of the enzyme complexed with both the NADPH cofactor and zopolrestat, however, clearly shows the inhibitor occupying the region directly above the nicotinamide of the NADPH and, therefore, the active site (Figures 5, 6, and 7). 

Why do we need vitamins Early clues came in 1935 when nicotinamide was found in NAD+ by H. von Euler and associates and in NADP+ by Warburg and Christian. Two years later, K. Lohman and P. Schuster isolated pure cocarboxylase, a dialyz-able material required for decarboxylation of pyruvate by an enzyme from yeast. It was shown to be thiamin diphosphate (Fig. 15-3). Most of the water-soluble vitamins are converted into coenzymes or are covalently bound into active sites of enzymes. Some lipid-soluble vitamins have similar functions but others, such as vitamin D and some metabolites of vitamin A, act more like hormones, binding to receptors that control gene expression or other aspects of metabolism. 

Functioning of the enzyme requires the presence of a coenzyme, nicotinamide adenine dinucleotide which exists in its oxidized (NAD+) or reduced (NADH) forms. The structure of NADH is shown in (177). Reduction or oxidation occurs by transfer of the pro-R C-4 hydrogen atom of the nicotinamide stereospecifically to or from the substrate. The reaction is therefore a ternary one, with the substrate and coenzyme necessarily within the active site for the reaction to occur.l46Sa... 

The 2.9 A resolution structure reveals that the NADH is bound in the enzyme binding domains by an extensive series of hydrogen bonds through the oxygen atoms of the pyrophosphate and the amide group of the nicotinamide. However, no atom in the coenzyme molecule is within bonding distance of the active site zinc atom, the closest being 4.5 A from the metal ion. 

A reversible covalent modification that plants use extensively is the reduction of cystine disulfide bridges to sulf-hydryls. Many of the enzymes of photosynthetic carbohydrate synthesis are activated in this way (table 9.3). Some of the enzymes of carbohydrate breakdown are inactivated by the same mechanism. The reductant is a small protein called thioredoxin, which undergoes a complementary oxidation of cysteine residues to cystine (fig. 9.5). Thioredoxin itself is reduced by electron-transfer reactions driven by sunlight, which serves as a signal to switch carbohydrate metabolism from carbohydrate breakdown to synthesis. In one of the regulated enzymes, phosphoribulokinase, one of the freed cysteines probably forms part of the catalytic active site. In nicotinamide-adenine dinucleotide phosphate (NADP)-malate dehydrogenase and fructose-1,6-bis-... 

Many other redox reactions are potentially amenable to antibody catalysis. For example, the chemistry of the P-450 cytochromes, including the hydroxylation of alkanes and the epoxidation of alkenes, can be mimicked with synthetic porphyrins. Incorporation of such molecules into antibody active sites could conceivably yield new catalysts that combine the intrinsic reactivity of the cofactor with the tailored selectivity of the binding pocket. Work is just beginning in this area, but preliminary studies with porphyrin haptens have yielded some interesting results.126-130 Novel redox chemistry can also be anticipated for antibodies containing metal ions, flavins, nicotinamide analogs, and other reactive moieties. 

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. 

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