Nicotinamide nucleotide binding

Boger m) showed that the transhydrogenase activity catalyzed by ferredoxin-NADP reductase obtained from Bumilleriopsis filiformis, which is very similar to the spinach enzyme, is regulated by ferredoxin and that one common nicotinamide nucleotide binding site is involved in both the diaphorase and the transhydrogenase reactions. 

It is important to recognize similarities in functional sites. However, differences are also important as they are often related to specificity. Many protein families share details of molecular function but vary in finer details such as their substrate specificity. Therefore those residues that are involved in discerning subclasses are likewise important in defining functional sites (Hannenhalli and Russell, 2000). For examples, the nicotinamide nucleotide binding core, (a2p3)2 are common to dehydrogenases but the key residue, Asp/Glu for NAD is replaced by Arg for NADP dependent enzymes. Similarly, key catalytic residues are common to all protein kinases but two regions of the sequence differ and are known to confer the specificity for either Ser/Thr or Tyr specific enzymes. 

NADPH/NADP+, reduced/oxidized nicotinamide adenine dinucleotide phosphate Nase, nucleotidase NBD, nucleotide-binding domain of ABC-TR... 

Although the nicotinamide nucleotide coenzymes function in a large number of oxidation and reduction reactions, thus carmot be exploited as a means of assessing the state of the body s niacin reserves, because the coenzymes are not firmly attached to their apoenzymes, as are coenzymes derived from thiamin (Section 6.5.3), riboflavin (Section 7.5.3), and vitamin Be (Section 9.5.3), but act as cosubstrates of the reactions, binding to and leaving the enzyme as... 

Nicotinamide-nucleotide-linked dehydrogenases were among the earliest two-substrate enzymes to be subjected to detailed kinetic study by steady-state 1-3) and rapid reaction techniques (4), and provided much of the original stimulus for the necessary extension of kinetic theory already developed for one-substrate and hydrolytic enzymes S-8). This was partly because of the convenience and precision with which rates can be measured by means of the light absorption or fluorescence emission 9-11) of the reduced coenzymes and because of the changes of these properties which accompany the binding of reduced coenzymes to many dehydrogenases 12,13). 

Some compounds that were historically considered as coenzymes do not remain bound to the active site of the enzyme, but bind and leave in the same way as other substrates. Such compounds include the nicotinamide nucleotide coenzymes (NAD and NADP) and coenzyme A. Although they are not really coenzymes, they are present in the cell in very much smaller concentrations than most substrates, and are involved in a relatively large number of reactions, so that they turn over rapidly. 

The vitamin niacin (section 11.8) is important for the formation of two closely related compounds, the nicotinamide nucleotide coenzymes — nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). As shown in Figure 2.16, they differ only in that NADP has an additional phosphate group attached to the ribose. The whole of the coenzyme molecule is essential for binding to enzymes, and most enzymes can bind and use only one of these two coenzymes, either NAD or NADP, despite the overall similarity in their structures. 

Unlike flavins and metal coenzymes, the nicotinamide nucleotide coenzymes do not remain bound to the enzyme, but act as substrates, binding to the enzyme, undergoing reduction and then leaving. The reduced coenzyme is then reoxidized either by reaction with another enzyme, for which it acts as a hydrogen donor, or by way of the mitochondrial electron transport chain (section 3.3.1.2). Cells contain only a small amount of NAD(P) (of the order of400 nmol/g in liver), which is rapidly cycled between the oxidized and reduced forms by different enzymes. 

Fig. 3. The "Rossmann" fold of EF-Tu (Rubin et al., 1981) as compared to the NAD binding domain of lactate dehydrogenase (LDH) (Rossmann et al., 1975). NAD is a dinucleotide and consequently the nucleotide binding fold is repeated. A and N represent the locations of the adenine and nicotinamide moieties. EF-Tu binds GDP or GTP (indicated with a G) at the standard location of the nucleotide binding fold. Only one of these structures is formed in EF-Tu.

Pantothenic acid, sometimes called vitamin B3, is a vitamin that makes up one part of a complex coenzyme called coenzyme A (CoA) (Figure 18.23). Pantothenic acid is also a constituent of acyl carrier proteins. Coenzyme A consists of 3, 5 -adenosine bisphosphate joined to 4-phosphopantetheine in a phosphoric anhydride linkage. Phosphopantetheine in turn consists of three parts /3-mercaptoethylamine linked to /3-alanine, which makes an amide bond with a branched-chain dihydroxy acid. As was the case for the nicotinamide and flavin coenzymes, the adenine nucleotide moiety of CoA acts as a recognition site, increasing the affinity and specificity of CoA binding to its enzymes. 

Glucose dehydrogenase (GDH) is a key initial enzyme in the energy production process that uses nucleotide cofactors to activate monosaccharide sugars as a prelude to their subsequent breakdown into pyruvate to enter the Krebs cycle. Nicotinamide adenine dinucleotide phosphate (NADP+, 765 Da) is the preferred cofactor, nicotinamide adenine dinucleotide (NAD", 663 Da) will act as a lower activity cofactor, and flavine adenine dinucleotide (FAD, 830 Da) will not bind nor act as cofactor. Preferred monosaccharide substrates for the enzyme are glucose and galactose (180 Da). Other monosaccharides (e.g. fructose, 180 Da) and disaccharides (e.g. maltose, sucrose, 342 Da) cannot act as substrates. 

In this section we shall consider only the coenzyme active site the regulatory sites for purine nucleotides will be discussed later. Cross and Fisher [309] have divided the coenzyme site into two subsites, one specific for the amide portion of the nicotinamide and the other for the adenosine diphosphate moiety. Binding of coenzyme at the amide subsite causes perturbations in the tyrosine and tryptophan absorption regions. Presumably, the tryptophan involved is the one found by Cross et al. (SOI) to have a red-shifted spectrum upon coenzyme binding. 

There are specific binding sites for the adenine nucleotide portion of the coenzyme (shown in red to the right of the dashed line) and for the nicotinamide portion of the coenzyme (shown in yellow to the left of the dashed line), in addition to the binding site for the substrate. Specific interactions with the enzyme hold the substrate and coenzyme in the proper positions. Sites of interaction are shown as a series of pale green lines. 

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