NADPH and NADP

Indicators There are certain compounds that are suitable as indicators for sensitive and specific clinical analysis. Nicotinamide adenine dinucleotide (NAD) occurs in oxidized (NAD" ) and reduced (NADH) forms. Nicotinamide adenine dinucleotide phosphate (NADP) also has two states, NADP" and NADPH. NADH has a very high uv—vis absorption at 339 nm, extinction coefficient = 6300 (M cm) , but NAD" does not. Similarly, NADPH absorbs light very strongly whereas NADP" does not. 

Figure 7-9. Absorption spectra of NAD and NADH. Densities are for a 44 mg/L solution in a cell with a 1 cm light path. NADP and NADPH have spectrums analogous to NAD and NADH, respectively.

The role of NADPH as protective donor 58, 59) seems to correlate well with the tendency of the different categories of catalases to form the inactive compoimd II. Only those classical catalases that are vulnerable to the latter inactivation step show NADP+ and NADPH binding. The scheme shown in Fig. 3B summarizes the minimal revisions of the 1958 scheme (Fig. 3A) needed to accommodate the newer findings. 

Reduced forms of these coenzymes absorb ultraviolet light near 340 nm, whereas the oxidized forms do not. For NAD at neutral pH, the maximal absorbance band (s = 18000 M cm ) occurs at 260 nm another absorbance band (s = 8000 M cm ) occurs at 230 nm. For NADH, the maximal absorbance band (s = 16900 M cm ) occurs at 259 nm a second absorbance band (s = 6220 M cm ) occurs at 339 nm two weaker bands occur at 234 and 290 nm with respective s values of 6600 M cm and 1300 M cm h The same is true for NADP and NADPH at 339 nm. Occasionally, investigators have used thio-NADH which has a much stronger absorbance around 366 nm. See Absorption Spectroscopy (Fig. 4, pg. 5) Nicotinic Acid Analogs and Coenzymes... 

Entry of glucose 6-phosphate either into glycolysis or into the pentose phosphate pathway is largely determined by the relative concentrations of NADP+ and NADPH. 

The surface plasmon resonance minimum reflectivity in Figure 20-23 shifts by —0.15° when 1 mM NAD+ binds to the imprinted polymer. The shift is not as great for the related species NADH, NADP+, and NADPH, confirming that the imprinted polymer selectively binds NAD+. When the observed reflectivity was fitted to the theoretical response, the polymer film was calculated to be 22 3 nm thick and had a binding capacity of 2.26 xg NAD+/cm2. When 1 mM NAD+ binds to the polymer, the refractive index of the polymer layer changes from 1.45 to 1.40 and the layer thickness increases by 3.0 0.2 nm. 

Figure 15-2 Absorption spectra of NAD+ and NADH. Spectra of NADP+ and NADPH are nearly the same as these. The difference in absorbance between oxidized and reduced forms at 340 nm is the basis for what is probably the single most often used spectral measurement in biochemistry. Reduction of NAD+ or NADP+ or oxidation of NADH or NADPH is measured by changes in absorbance at 340 nm in many methods of enzyme assay. If a pyridine nucleotide is not a reactant for the enzyme being studied, a coupled assay is often possible. For example, the rate of enzymatic formation of ATP in a process can be measured by adding to the reaction mixture the following enzymes and substrates hexokinase + glucose + glucose-6-phosphate dehydrogenase + NADP+. As ATP is formed, it phosphorylates glucose via the action of hexokinase. NADP+ then oxidizes the glucose 6-phosphate that is formed with production of NADPH, whose rate of appearance is monitored at 340 nm.

Further evidence32 has been obtained which strongly supports the conclusion (c/. Vol. 10, p. 19) that geissoschizine (101) is at a shunt in the biosynthesis of the three tissue-culture alkaloids (102)—(104). Incubation of (101) with the enzyme preparation plus NADP+ and NADPH in D20 afforded a sample of (103) that contained one deuterium atom per molecule. Geissoschizine (101) must thus be involved in biosynthesis after (98). With NADPD as co-factor, a sample of (103) was isolated that contained a single deuterium atom, located at C-21, in each molecule. Moreover, geissoschizine with deuterium that was stereochemically a at C-21 gave (103) that was devoid of label. These two results support biosynthesis via (99). 

Aldehyde oxidase purified from maize coleoptiles is a multicomponent enzyme that contains a molybdenum cofactor, nonheme iron, and flavin adenine dinucleotide (FAD) as prosthetic groups.111 When substrate specificity of the aldehyde oxidase was tested, good activity was detected with IAAld, indole-3-aldehyde, and benzaldehyde among others. The addition of NADP and NADPH did not change the activity. In contrast, in maize endosperm, tryptophan-dependent IAA biosynthesis was dependent on an NADP/NADPH redox system, which may mean that the two tissues of maize are utilizing different pathways or different redox systems for IAA biosynthesis.112... 

As is indicated in Table 5-3, P680, P70o> the cytochromes, plastocyanin, and ferredoxin accept or donate only one electron per molecule. These electrons interact with NADP+ and the plastoquinones, both of which transfer two electrons at a time. The two electrons that reduce plastoquinone come sequentially from the same Photosystem II these two electrons can reduce the two >-hemes in the Cyt b(f complex, or a >-heme and the Rieske Fe-S protein, before sequentially going to the /-heme. The enzyme ferre-doxin-NADP+ oxidoreductase matches the one-electron chemistry of ferredoxin to the two-electron chemistry of NADP. Both the pyridine nucleotides and the plastoquinones are considerably more numerous than are other molecules involved with photosynthetic electron flow (Table 5-3), which has important implications for the electron transfer reactions. Moreover, NADP+ is soluble in aqueous solutions and so can diffuse to the ferredoxin-NADP+ oxidoreductase, where two electrons are transferred to it to yield NADPH (besides NADP+ and NADPH, ferredoxin and plastocyanin are also soluble in aqueous solutions). 

Since the chemical shifts of carbon nuclei in the nicotinamide moiety of the di-phosphopyridine nucleotides (NAD and NADH) are almost identical to the tri-phosphopyridine nucleotides (NADP and NADPH). the intensities of the oxidized (NAD ) and reduced (NADH) resonances determine a mean reduction charge of the cells defined by... 

As pointed out previously in this review the steady-state kinetics of mitochondrial transhydrogenase, earlier interpreted to indicate a ternary Theorell-Chance mechanism on the basis of competitive relationships between NAD and NADH and between NADP and NADPH, and noncompetitive relationships between NAD" and NADP" and between NADH and NADPH, has been reinterpreted in the light of more recent developments in the interpretation of steady-state kinetic data. Thus, although the product inhibition patterns obtained in the earlier reports [75-77] using submitochondrial particles were close to identical to those obtained in a more recent report [90] using purified and reconstituted transhydrogenase, the reinterpretation favors a random mechanism with the two dead-end complexes NAD E NADP and NADH E NADPH. A random mechanism is also supported by the observation that the transhydrogenase binds to immobilized NAD as well as NADP [105] in the absence of the second substrate. 

Niacin is a water-soluble vitamin. The RDA of niacin for the adult man is 19 mg. Niacin is converted in the bi>dy to the cofactor nicotinamide adenine dinucleotide (NAD). NAD also exists in a phosphorylated form, NADP The phosphate group occurs on the 2-hydrr>xyl group of the AMP half of the coenzyme, NAD and NADP are used in the catalysis of oxidation and reduction reactions. These reactions are called redox reactions. NAD cycles between the oxidized form, NAD, and the reduced form, NADH + H. The coenzyme functions to accept and donate electrons. NADP behaves in a similar fashion. It occurs as NADP and NADPH + HT The utilization of NAD is illustrated in the sections on glycolysis, the malatc-aspartate shuttle, ketone body metabolism, and fatty acid oxidation. The utilization of NADP is illustrated in the sectirrns concerning fatty acid synthesis and the pentose phosphate pathway. 

Phosphate, a common component of enzymatic systems and an Integral part of NADP and NADPH, is a particularly effective acid catalyst for this reaction. 

Figure 2. Cycles of life and death. A brief overview showing how paraquat (and similarly diquat) interacts with two of the most fundamental processes of life photosynthesis in the chloroplast and respiration in the mitochondrion. Abbreviations ADP, adenosine diphosphate ATP, adenosine triphosphate e, electron proton NAD and NADH, oxidised and reduced forms of nicotinamide adenine dinucleotide NADP and NADPH, oxidised and reduced forms of nicotinamide adenine dinucleotide phosphate Paraquat, paraquat radical Pi, inorganic phosphate superoxide radical.

In many of the examples cited of affinity labeling of enzymes by periodate-oxidized nucleotides, it has been assumed that the reaction involved formation of a Schiff base with an enzymic lysine, as in Fig. 3a,b. However, in very few papers has direct evidence been presented supporting the existence of a Schiff base intermediate. Lowe and Beechey (83), after examining in detail the structure of periodate-oxidized ATP, concluded that in aqueous solution there is little free aldehyde rather, the compound exists predominantly as an equilibrium mixture of three dialdehyde monohydrates (cyclic hemiacetals) and a dihydrate. The presence of cyclic hemiacetals may account for the ability of periodate-oxidized NADP and NADPH to function as coenzymes in several enzymic reactions (e.g., 78, 81). In many cases, the product of the covalent reaction of an enzyme and periodate-oxidized nucleotide may be a dihydroxymorpholino derivative (Fig. 3c), which is similar to the cyclic hemiacetals observed in aqueous solu-... 

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. 

The differentiation between the coenzymes used in catabolism and anabolism is maintained because the enzymes that catalyze these oxidation-reduction reactions exhibit strong specificity for a particular coenzyme. For example, an enzyme that catalyzes an oxidation reaction can readily tell the difference between NAD and NADP if the enzyme is in a catabolic pathway, it will bind NAD, but not NADP. In addition, the relative concentrations of the coenzymes in the cell encourage binding of the appropriate coenzyme. For example, because NAD and NADH are catabolic coenzymes and catabolic reactions are most often oxidation reactions, the NAD concentration in the cell is much greater than the NADH concentration. (The cell maintains its [NAD" "]/[NADH] ratio near 1000.) Because NADP" and NADPH are anabolic coenzymes and anabolic pathways are predominantly reduction reactions, the concentration of NADPH in the cell is greater than the concentration of NADP" ". (The ratio of [NADP" "]/[NADPH] is maintained at about 0.01.)... 

Ziegler, G.A. and G.E. Schulz (2000). Crystal structures of adrenodoxin reduetase in eomplex with NADP(+) and NADPH suggesting a mechanism for the electron transfer of an enzyme family. Biochemistry 39, 10986-10995. 

The structure of this coenzyme is shown in Figure 2.2. It is made up of nicotinamide, ribose, phosphate and the nucleotide adenine. It exists in two forms, as shown. These differ in their oxidation state. NADP contains a pyridinium ring while NADPH contains a dihydropyridine. They can be interconverted by transfer of a hydride anion. The related coenzymes NAD and NADH are the same as NADP and NADPH except that they lack the phosphate ester at the 2 position (i.e. the... 

Remember, in equilibrium constants the molar concentrations of the reactants are raised to a power equal to the number of moles taking part in the reaction. Therefore, in this problem the [GSH] is squared because, for each mole of GSSG, NADP+, and NADPH, two moles of GSH are involved. 

EXAMPLE 11.20 How do NADP+ and NADPH differ from NAD+ and NADH ... 

Red cell NADP+ and NADPH in glucose-6-phosphate dehydrogenase deficiency. 

Figure 2 Absorbance of NAD and NADH as a function of wavelength. NADP and NADPH show nearly the same curves,...

The desaturase (and following cyclase) enzymes are integral membrane proteins that can be solubilized by the detergent CHAPS. Enzymatic activity is lost when the enzymes are solubilized, but it is restored when reconstituted in proteoliposomes (Britton, 1993). In the first phase of desaturation, the conversion of (15Z)-phytoene into (15Z)- -carotene occurred in the dark, with oxygen as an essential requirement. The reaction was independent of cofactors such as NADP and NADPH. (15Z)- -Carotene could not be desaturated further, but when this compound was isomerized by light, the isomeric mixture formed underwent further desaturation to produce lycopene, not... 

NADP /NADPH is the major redox molecular pair in living organisms. Remember this symbol NADP and NADPH. The oxidoreductive reaction given... 

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