Nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide is a coenzyme central to metabolism. Found in all living cells, NAD is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine nucleobase and the other, nicotinamide. NAD exists in two forms: an oxidized and reduced form, abbreviated as NAD and NADH, respectively.
In cellular metabolism, NAD is involved in redox reactions, carrying electrons from one reaction to another, so it is found in two forms: NAD is an oxidizing agent, accepting electrons from other molecules and becoming reduced; with H+, this reaction forms NADH, which can be used as a reducing agent to donate electrons. These electron transfer reactions are the main function of NAD. It is also used in other cellular processes, most notably as a substrate of enzymes in adding or removing chemical groups to or from proteins, in posttranslational modifications. Because of the importance of these functions, the enzymes involved in NAD metabolism are targets for drug discovery.
In organisms, NAD can be synthesized from simple building-blocks from either tryptophan or aspartic acid, each a case of an amino acid. Alternatively, more complex components of the coenzymes are taken up from nutritive compounds such as vitamin B3. Similar compounds are produced by reactions that break down the structure of NAD, providing a salvage pathway that recycles them back into their respective active form.
In the name NAD, the superscripted plus sign indicates the positive formal charge on one of its nitrogen atoms.
Some NAD is converted into the coenzyme nicotinamide adenine dinucleotide phosphate, whose chemistry largely parallels that of NAD, though its predominant role is as a coenzyme in anabolic metabolism.
NADP is a reducing agent in anabolic reactions like the Calvin cycle and lipid and nucleic acid syntheses. NADP exists in two forms: NADP+, the oxidized form, and NADPH, the reduced form. NADP is similar to nicotinamide adenine dinucleotide, but NADP has a phosphate group at the C-2′ position of the adenosyl.
Physical and chemical properties
Nicotinamide adenine dinucleotide consists of two nucleosides joined by pyrophosphate. The nucleosides each contain a ribose ring, one with adenine attached to the first carbon atom and the other with nicotinamide at this position.The compound accepts or donates the equivalent of H−. Such reactions involve the removal of two hydrogen atoms from a reactant, in the form of a hydride ion, and a proton. The proton is released into solution, while the reductant RH is oxidized and NAD reduced to NADH by transfer of the hydride to the nicotinamide ring.
From the electron pair of the hydride ion, one electron is attracted to the slightly more electronegative atom of the nicotinamide ring of NAD, becoming part of the nicotinamide moiety. The remaining hydrogen atom is transferred to the carbon atom opposite the N atom. The midpoint potential of the NAD/NADH redox pair is −0.32 volts, which makes NADH a moderately strong reducing agent. The reaction is easily reversible, when NADH reduces another molecule and is re-oxidized to NAD. This means the coenzyme can continuously cycle between the NAD and NADH forms without being consumed.
In appearance, all forms of this coenzyme are white amorphous powders that are hygroscopic and highly water-soluble. The solids are stable if stored dry and in the dark. Solutions of NAD are colorless and stable for about a week at 4 °C and neutral pH, but decompose rapidly in acidic or alkaline solutions. Upon decomposition, they form products that are enzyme inhibitors.
Both NAD and NADH strongly absorb ultraviolet light because of the adenine. For example, peak absorption of NAD is at a wavelength of 259 nanometers, with an extinction coefficient of 16,900 M−1cm−1. NADH also absorbs at higher wavelengths, with a second peak in UV absorption at 339 nm with an extinction coefficient of 6,220 M−1cm−1. This difference in the ultraviolet absorption spectra between the oxidized and reduced forms of the coenzymes at higher wavelengths makes it simple to measure the conversion of one to another in enzyme assays – by measuring the amount of UV absorption at 340 nm using a spectrophotometer.
NAD and NADH also differ in their fluorescence. Freely diffusing NADH in aqueous solution, when excited at the nicotinamide absorbance of ~335 nm, fluoresces at 445–460 nm with a fluorescence lifetime of 0.4 nanoseconds, while NAD does not fluoresce. The properties of the fluorescence signal changes when NADH binds to proteins, so these changes can be used to measure dissociation constants, which are useful in the study of enzyme kinetics. These changes in fluorescence are also used to measure changes in the redox state of living cells, through fluorescence microscopy.
NADH can be converted to NAD+ in a reaction catalysed by copper, which requires hydrogen peroxide. Thus, the supply of NAD+ in cells requires mitochondrial copper.
Concentration and state in cells
In rat liver, the total amount of NAD and NADH is approximately 1 μmole per gram of wet weight, about 10 times the concentration of NADP and NADPH in the same cells. The actual concentration of NAD in cell cytosol is harder to measure, with recent estimates in animal cells ranging around 0.3 mM, and approximately 1.0 to 2.0 mM in yeast. However, more than 80% of NADH fluorescence in mitochondria is from bound form, so the concentration in solution is much lower.NAD concentrations are highest in the mitochondria, constituting 40% to 70% of the total cellular NAD. NAD in the cytosol is carried into the mitochondrion by a specific membrane transport protein, since the coenzyme cannot diffuse across membranes. The intracellular half-life of NAD+ was claimed to be between 1–2 hours by one review, whereas another review gave varying estimates based on compartment: intracellular 1–4 hours, cytoplasmic 2 hours, and mitochondrial 4–6 hours.
The balance between the oxidized and reduced forms of nicotinamide adenine dinucleotide is called the NAD/NADH ratio. This ratio is an important component of what is called the redox state of a cell, a measurement that reflects both the metabolic activities and the health of cells. The effects of the NAD/NADH ratio are complex, controlling the activity of several key enzymes, including glyceraldehyde 3-phosphate dehydrogenase and pyruvate dehydrogenase. In healthy mammalian tissues, estimates of the ratio of free NAD to NADH in the cytoplasm typically lie around 700:1; the ratio is thus favorable for oxidative reactions. The ratio of total NAD/NADH is much lower, with estimates ranging from 3–10 in mammals. In contrast, the NADP/NADPH ratio is normally about 0.005, so NADPH is the dominant form of this coenzyme. These different ratios are key to the different metabolic roles of NADH and NADPH.
Biosynthesis
NAD is synthesized through two metabolic pathways. It is produced either in a de novo pathway from amino acids or in salvage pathways by recycling preformed components such as nicotinamide back to NAD. Although most tissues synthesize NAD by the salvage pathway in mammals, much more de novo synthesis occurs in the liver from tryptophan, and in the kidney and macrophages from nicotinic acid.''De novo'' production
Most organisms synthesize NAD from simple components. The specific set of reactions differs among organisms, but a common feature is the generation of quinolinic acid from an amino acideither tryptophan in animals and some bacteria, or aspartic acid in some bacteria and plants. The quinolinic acid is converted to nicotinic acid mononucleotide by transfer of a phosphoribose moiety. An adenylate moiety is then transferred to form nicotinic acid adenine dinucleotide. Finally, the nicotinic acid moiety in NaAD is amidated to a nicotinamide moiety, forming nicotinamide adenine dinucleotide.In a further step, some NAD is converted into NADP by NAD kinase, which phosphorylates NAD. In most organisms, this enzyme uses adenosine triphosphate as the source of the phosphate group, although several bacteria such as Mycobacterium tuberculosis and a hyperthermophilic archaeon Pyrococcus horikoshii, use inorganic polyphosphate as an alternative phosphoryl donor.
Salvage pathways
Despite the presence of the de novo pathway, the salvage reactions are essential in humans; a lack of vitamin B3 in the diet causes the vitamin deficiency disease pellagra. This high requirement for NAD results from the constant consumption of the coenzyme in reactions such as posttranslational modifications, since the cycling of NAD between oxidized and reduced forms in redox reactions does not change the overall levels of the coenzyme.The major source of NAD in mammals is the salvage pathway which recycles the nicotinamide produced by enzymes utilizing NAD. The first step, and the rate-limiting enzyme in the salvage pathway is nicotinamide phosphoribosyltransferase, which produces nicotinamide mononucleotide. NMN is the immediate precursor to NAD+ in the salvage pathway.
Besides assembling NAD de novo from simple amino acid precursors, cells also salvage preformed compounds containing a pyridine base. The three vitamin precursors used in these salvage metabolic pathways are nicotinic acid, nicotinamide and nicotinamide riboside. These compounds can be taken up from the diet and are termed vitamin B or niacin. However, these compounds are also produced within cells and by digestion of cellular NAD. Some of the enzymes involved in these salvage pathways appear to be concentrated in the cell nucleus, which may compensate for the high level of reactions that consume NAD in this organelle. There are some reports that mammalian cells can take up extracellular NAD from their surroundings, and both nicotinamide and nicotinamide riboside can be absorbed from the gut.
The salvage pathways used in microorganisms differ from those of mammals. Some pathogens, such as the yeast Candida glabrata and the bacterium Haemophilus influenzae are NAD auxotrophs – they cannot synthesize NAD – but possess salvage pathways and thus are dependent on external sources of NAD or its precursors. Even more surprising is the intracellular pathogen Chlamydia trachomatis, which lacks recognizable candidates for any genes involved in the biosynthesis or salvage of both NAD and NADP, and must acquire these coenzymes from its host.