NADP+, NADPH structure

The coenzyme NADP+ differs from NAD+ only by the presence of a phosphate group (-P04=)on one of the ribose units (Figure 13.4). This seemingly small change in structure allows NADP+ to interact with NADP+-specific enzymes that have unique roles in the cell. For example, the steady-state ratio of NADP+/NADPH in the cytosol of hepato-cytes is approximately 0.1, which favors the use of NADPH in reductive biosynthetic reactions. This contrasts with the high ratio of NAD7NADH (approximately 1000 in the cytosol of hepatocytes), which favors an oxidative role for NAD+. This section summarizes some important NADP+ or NADPH-specific functions. 

The 1.25 A-crystal structure of the mouse SR in complex with NADP has been solved. The 261 amino acids of the monomer fold into a single domain a//3-structure. A seven-stranded parallel /3-sheet in the center of the molecule is sandwiched by two arrays of three a-helices. The association of two monomers to the active homodimeric SR leads to the formation of a four-helix bundle (Figure 13). Owing to the two-folded crystallographic symmetry of the homodimeric molecule, the parallel /3-sheets in monomer A is in an antiparallel orientation relative to the /3-sheet of monomer B enclosing an angle of 90°. The overall dimensions of the SR dimer are 40 A X 50 A x 80 A. The two substrate pockets bind sepiapterin (or 6-pyruvoyl-tetrahydropterin 42), and the cofactor NADP/NADPH from opposite sides to the enzyme. 

The only way in which NADP differs structurally from NAD is in the phosphate group bonded to the 2 -OH group of the ribose of the adenine nucleotide this explains the addition of P to the name. NAD and NADH are generally used as coenzymes in catabolic reactions NADP and NADPH are generally used as coenzymes in anabolic reactions. 

FIGURE 9.9. Structures of the NAD /NADH and NADP /NADPH coenzyme redox couples. 

Nicotinamide adenine dinucleotide (NAD ) and nicotinamide adenine dinucleotide phosphate (NADP+) are structurally dinucleotides (Fig. 29). The two substances differ from each other by one phosphate residue, which is present in NADP" " and is attached to the 2 hydroxyl group of one of the ribose residues. The actual redox system is the nicotinamide which can be reversibly reduced, again by a 2 electron transition. Flavoproteins are very often coupled with NAD+or NADP+. In photosynthesis reduced FAD can transmit 2H (= 2e -)- 2H+) to NADP+, leading to NADPH -I- H+. 

Nasr et failed to observe a change in the ratio of NADPH to NADP in the tracheal epithelium of rats exposed to ozone at 33 ppm for an hour. This apparently negative in vivo finding is not surprising, inasmuch as NADP will be rapidly reduced back to NADPH if ozone does not disrupt the structural integrity of pyridine nucleotides. In addition, de novo thesis of pyridine nucleotides may also occur. The intracellular ratio of reduced to oxidized pyridine nucleotides is under tine cellular control, in that the oxidation of NADPH or NADH results in the stimulation of enzymatic activity, which restores the initial ratio. In the case of NADPH, its oxidation increases the activity of the hexose monophosphate shunt this also occurs after the oxidation of glutathione. The rel-... 

The pyridine nucleotides NAD and NADP always function in unbound form. The oxidized forms contain an aromatic nicotinamide ring in which the positive charge is delocalized. The right-hand example of the two resonance structures shown contains an electron-poor, positively charged C atom at the para position to nitrogen. If a hydride ion is added at this point (see above), the reduced forms NADH or NADPH arise. No radical intermediate steps occur. Because a proton is released at the same time, the reduced pyridine nucleotide coenzymes are correctly expressed as NAD(P)H+HT... 

Transhydrogenases function in a similar way within bacteria. Whether from E. coli, photosynthetic bacteria, or bovine mitochondria, transhydrogenases have similar structures.285 Two 510-residue a subunits associate with two 462-residue P subunits to form an OC2P2 tetramer with 10-14 predicted transmembrane helices. Tire a subunits contain separate NAD(H) and NADP(H) binding sites. A conformational change appears to be associated with the binding or release of the NADP+ or NADPH 287... 

Mesophyll cells use C02 from the air to convert phospho-enolpyruvate to oxaloacetate (fig. 15.28). Oxaloacetate is reduced to malate, which then moves to the bundle sheath cells that surround the vascular structures in the interior of the leaf. Here malate is decarboxylated to pyruvate in an oxidative reaction that reduces NADP+ to NADPH. The pyruvate returns to the mesophyll cells, where it is phos-phorylated to phosphoenolpyruvate. This phosphorylation is driven by splitting of ATP to AMP and pyrophosphate and subsequent hydrolysis of the pyrophosphate to phosphate. 

An important aspect of enzymatic oxidation-reduction reactions involves the transfer of hydrogen atoms. This transfer is mediated by coenzymes (substances that act together with enzymes) nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). These two species pick up H atoms to produce NADH and NADPH, respectively, both of which can function as hydrogen atom donors. Another pair of species involved in oxidation-reduction processes by hydrogen atom transfer consists of flavin adenine triphosphate (FAD) and its hydrogenated form FADH2. The structural formulas of NAD and its cationic form, NAD+, are shown in Figure 4.7. 

The crystal structures of the E. coli DHFR-methotrexate binary complex (Bolin et al., 1982), of the Lactobacillus casei (DHFR-NADPH-methotrexate ternary complex (Filman et al., 1982), of the human DHFR-folate binary complex (Oefner et al., 1988), and of the mouse (DHFR-NADPH-trimethoprim tertiary complex (Stammers et al., 1987) have been resolved at a resolution of 2 A or better. The crystal structures of the mouse DHFR-NADPH-methotrexate (Stammers et al., 1987) and the avian DHFR—phenyltriazine (Volz et al., 1982) complexes were determined at resolutions of 2.5 and 2.9 A, respectively. Recently, the crystal structure of the E. coli DHFR—NADP + binary and DHFR-NADP+-folate tertiary complexes were resolved at resolutions of 2.4 and 2.5 A, respectively (Bystroff et al., 1990). DHFR is therefore the first dehydrogenase system for which so many structures of different complexes have been resolved. Despite less than 30% homology between the amino acid sequences of the E. coli and the L. casei enzymes, the two backbone structures are similar. When the coordinates of 142 a-carbon atoms (out of 159) of E. coli DHFR are matched to equivalent carbons of the L. casei enzyme, the root-mean-square deviation is only 1.07 A (Bolin et al., 1982). Not only are the three-dimensional structures of DHFRs from different sources similar, but, as we shall see later, the overall kinetic schemes for E. coli (Fierke et al., 1987), L. casei (Andrews et al., 1989), and mouse (Thillet et al., 1990) DHFRs have been determined and are also similar. That the structural properties of DHFRs from different sources are very similar, in spite of the considerable differences in their sequences, suggests that in the absence, so far, of structural information for ADHFR it is possible to assume, at least as a first approximation, that the a-carbon chain of the halophilic enzyme will not deviate considerably from those of the nonhalophilic ones. 

Niacin ia a nutritional term applied to both nicotinic acid and nicotinamide and to a mixture of the two. Their structures and those of their coenzymes are given in Table 6.1. Numerous redox reactions use NAD+ and NADP+ or NADH and NADPH. The latter are used largely in reactions designed to reductively synthesize various substances, mostly in the extramitochondrial areas of the cell. NAD+, on the other hand, is used largely in its oxidized form in catabolic redox reactions. The rat liver cytosol NADPH/NADP+ ratio is about 80, whereas its NADH/NAD+ ratio is only 8 x 10 4. Table 6.3 lists some biochemical reactions in which these cofactors participate. It shows that they are of crucial importance in the metabolism of carbohydrates, fats, and amino acids. 

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