Nicotinamide adenine dinucleotide

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Nicotinamide adenine dinucleotide
Identifiers
CAS number	53-84-9
PubChem	925
MeSH	Nicotinamide-Adenine+Dinucleotide
Properties
Molecular formula	C21H27N7O14P2
Molar mass	663.425
Hazards
Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa) Infobox disclaimer and references

Nicotinamide adenine dinucleotide (NAD⁺ or in older notation DPN⁺) is an important coenzyme found in cells. It plays key biochemical roles as a carrier of electrons and a participant in metabolic redox reactions, as well as in cell signaling.^[1][2] There are two forms of this coenzyme in cells, NAD⁺ and the phosphorylated form NADP⁺. These two related coenzymes have similar chemistry, but perform different roles in metabolism. When both coenzymes are being discussed, they are referred to collectively as NAD(P)⁺.

NADH is the reduced form of NAD⁺, and NADPH is the reduced form of NADP⁺. In metabolism, NAD⁺ and NADP⁺ are used as oxidizing agents - they accept electrons from other molecules, whereas NADH and NADPH are reducing agents that donate electrons. In chemical terms, these coenzymes are reactants in hydride transfer reactions, which is the primary function of NAD(P)⁺.^{[citation needed]} However, they are also used in a range of cellular processes that do not involve redox reactions, such as second messenger systems and posttranslational modifications.

NAD⁺ can be synthesised from amino acids such as tryptophan. Alternatively, components of the coenzyme taken up in the diet or released by reactions that break down the structure of NAD⁺ can be salvaged and then recycled back into the active form.

Due to the central role of NAD(P)⁺ in metabolism, the enzymes involved in making and utilising these coenzymes have been described as promising targets for drug discovery.^[3]

[edit] Physical and chemical properties

Nicotinamide adenine dinucleotide consists of two ribose rings, one with adenine attached to its 1' carbon atom and the other with nicotinamide at this position; these two sugar-heterocycle moieties are joined together by a bridge of two phosphate groups through the 5' carbons. In NADP⁺, the ribose ring attached to the adenine has an additional phosphate group at the 2' position.

In appearance, both coenzymes are white amorphous hygroscopic powders that are highly water-soluble.^[4] The solids are stable if stored dry and in the dark. Solutions of NAD(P)⁺ are colorless and stable for about a week at 4°C and neutral pH, but decompose rapidly in acids. Upon decomposition, they form products that are enzyme inhibitors.^[5]

The reduction of NAD(P)⁺ to NAD(P)H.

NAD(P)⁺ absorbs strongly in the ultraviolet due to the adenine base. Peak absorption is at 259 nm, with an extinction coefficient of 16,900 M^-1 cm^-1. The reduced forms of NAD(P)H also absorb at a higher wavelength, with a second peak in UV absorption at 339 nm that has an extinction coefficient of 6,220 M^-1 cm^-1.^[6] This difference in the ultraviolet absorption spectra between the oxidised and reduced forms of the coenzymes makes it simple to measure the conversion of one to another in enzyme assays - by measuring the amount of UV absorption at 339 nm using a spectrophotometer.^{[citation needed]}

Cells use NAD(P)⁺ to accept or donate electrons in redox reactions. Such reactions (summarised below) involve the removal of two hydrogen atoms in the form of a hydride ion and a proton (H⁺) from the reactant (R). The proton is released into solution, while the reductant RH₂ is oxidised and NAD(P)⁺ reduced to NAD(P)H.

RH₂ + NAD(P)⁺ → NAD(P)H + H⁺ + R

From the hydride electron pair, one electron is transferred to the positively-charged nitrogen of the nicotinamide ring of NAD(P)⁺, and the second hydrogen atom transferred to the carbon atom opposite this nitrogen. The reaction is easily reversible, permitting the recycling of the coenzymes without depletion.

Ball-and-stick model of NAD+

Ball-and-stick model of NADH

Space-filling model of NADH

[edit] Binding to proteins

An example of the Rossmann fold, a structural domain of a decarboxylase protein from the bacterium Staphylococcus epidermidis (PDB ID 1G5Q) with the bound flavin mononucleotide cofactor shown.

Further information: Protein structure and Oxidoreductases

When bound to a protein, NAD(P)⁺ and NAD(P)H bind to a structural motif named the Rossmann fold.^[7] The motif is named after Michael Rossmann who was the first scientist to notice that this structure was a frequently-occurring motif in nucleotide binding proteins.^[8]This motif is found in many proteins that bind nucleotides, and is composed of three or more parallel beta strands linked by two alpha helices in the topological order beta-alpha-beta-alpha-beta. Because each Rossmann fold binds one nucleotide, binding domains for the dinucleotide NAD⁺ consist of two paired Rossmann folds, with each fold binding one nucleotide moiety of the cofactor.^{[citation needed]} Single Rossmann folds are found in proteins that bind mononucleotides, such as the cofactor FMN. However, the Rossman fold is not universal among NAD(P)-dependent enzymes, since a class of bacterial enzymes have recently been discovered that bind NAD(P)+ but lack Rossmann folds.^[9]

Despite this similarity in how proteins bind NAD⁺ and NADP⁺, enzymes almost always show a high level of specificity for either one or the other of these coenzymes.^[10] This specificity is related to the distinct metabolic roles of the two conezymes and results from distinct sets of amino acid residues in the coenzyme-binding pocket. For instance, in the active site of NADP-dependent enzymes, a specific ionic bond is formed between a basic amino acid side chain and the acidic phosphate group of NADP⁺. Conversely, in NAD-dependent enzymes the charge in this pocket is reversed, preventing NADP⁺ binding. There are a few exceptions to this general rule, and enzymes such aldose reductase, glucose-6-phosphate dehydrogenase, and methylenetetrahydrofolate reductase show dual-cofactor specificity in some species.^[11]

[edit] Biological concentration

In rat liver, the total amount of NAD⁺ and NADH is approximately 1 μmol^-1 g wet weight, about 10 times the concentration of NADP⁺ and NADPH in the same cells.^[12] The actual concentration of NAD⁺ in cell cytoplasm is hard to measure, with recent estimates in red blood cells ranging around 300 μM.^[13][14] However, over 80% of NADH is bound to proteins, so the concentration of free forms of the coenzymes is correspondingly much lower.^[15]

The balance between the oxidised and reduced forms of nicotinamide adenine dinucleotide is called the NAD⁺/NADH ratio. This ratio reflects the redox state of a cell, which affects both the metabolic activity and the health of that cell. The activity of several key enzymes is controlled by the NAD⁺/NADH ratio, including glyceraldehyde 3-phosphate dehydrogenase and pyruvate dehydrogenase.^[16] In healthy mammalian tissues, estimates of the NAD⁺/NADH ratio range around 1, so the concentrations of NAD⁺ and NADH are roughly comparable.^[16] In contrast, the NADP⁺/NADPH ratio is normally about 0.005, around 200 times lower than the NAD⁺/NADH ratio, so NADPH is by far the dominant form of this coenzyme.^[17] These different ratios reflect the different metabolic roles of NADH and NADPH.

[edit] Functions

Nicotinamide adenine dinucleotide is an essential part of metabolism.

[edit] Role in redox metabolism

A simplified outline of redox metabolism, showing how NAD⁺ and NADH link the citric acid cycle and oxidative phosphorylation.

Further information: Metabolism

The main role of NAD(P)⁺ in metabolism is the transfer of electrons from one redox reaction to another. This is most important in the release of energy from nutrients. Here, reduced compounds such as glucose are oxidized, thereby releasing energy. This energy is transferred to NAD⁺ by reduction to NADH, as part of glycolysis and the citric acid cycle. The NADH is then oxidized in turn by the electron transport chain, which pumps protons across a membrane and generates ATP through oxidative phosphorylation.^[18] Since both the oxidized and reduced forms of nicotinamide adenine dinucleotide are used in these linked sets of reactions, the cell maintains approximately equal concentrations of NAD⁺ and NADH; the high NAD⁺/NADH ratio allows this coenzyme to act as both an oxidizing and a reducing agent.^[19] In contrast, the phosphorylated form of this coenzyme functions primarily as a reducing agent in anabolism, so the NADP⁺/NADPH ratio is kept very low, allowing NADPH to drive redox reactions as a strong reducing agent.^[19]

Although it is most important in catabolism, NADH is also used in a few anabolic reactions, such as gluconeogenesis.^[20] This poses a problem for prokaryotes growing on nutrients that release only a small amount of energy upon oxidation. For example, nitrifying bacteria such as Nitrobacter oxidize nitrite to nitrate, donating the electrons to oxygen. The energy released in this reaction is enough to pump protons and generate ATP, but not enough to produce NADH directly.^[21] Since the cells still require NADH for anabolic reactions, they use a nitrite oxidoreductase to produce enough proton-motive force to run part of the electron transport chain in reverse, making it generate NADH.^[22][23]

[edit] Non-redox roles

The coenzyme NAD⁺ can also be consumed by in ADP-ribose transfer reactions. For example, some enzymes add the ADP-ribose moiety of this molecule to proteins, in a posttranslational modification called ADP-ribosylation.^[24] These reactions are involved in cell signaling and the control of many cell processes, including DNA repair and apoptosis.^[25] ADP-ribose can also be transferred to proteins in long branched chains, in a reaction called poly(ADP-ribosyl)ation.^[26] This protein modification is carried out by the poly ADP-ribose polymerases.^[26][27] The poly(ADP-ribose) structure is involved in the regulation of several cellular events and is most important in the cell nucleus, in processes such as DNA repair and telomere maintenance.^[27]

NAD⁺ can also be converted into cyclic ADP-ribose by ADP-ribosyl cyclases as part of a second messenger system.^[28] The cyclic ADP ribose second messenger molecule acts in calcium signaling by releasing calcium from intracellular stores.^[29] It does this by binding to and opening a class of calcium channels called ryanodine receptors.

Other NAD-dependent enzymes include bacterial DNA ligases, which join two DNA ends by using NAD as a substrate to donate an AMP group to the 5' phosphate of one DNA end. This intermediate is then attacked by the 3' hydroxyl group of the other DNA end, forming a new phosphodiester bond.^[30] This contrasts with eukaryotic DNA ligases, which use ATP to form the DNA-AMP intermediate. NAD⁺ is also consumed by sirtuins, which are NAD-dependent deacetylases, such as Sir2.^[31] These enzymes act by transferring an acetyl group from their substrate protein to the ADP-ribose moiety of NAD⁺; this cleaves the coenzyme and releases nicotinamide and O-acetyl-ADP-ribose. The sirtuins mainly seem to be involved in regulating transcription through deacetylating histones and altering nucleosome structure.^[32]

[edit] 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⁺.

[edit] De novo production

The two metabolic pathways that synthesize and consume NAD⁺. The abbreviations are defined in the text.

Most organisms can synthesize NAD(P) from simple components.^{[citation needed]} The specific set of reactions differs among organsisms, but a common feature is the generation of quinolinic acid (QA) from an amino acid - either tryptophan (Trp) in animals and some bacteria, or aspartic acid in some bacteria and plants.^[33] The quinolinic acid is converted to nicotinic acid mononucleotide (NaMN) by transfer of a phosphoribose moiety. An adenyl group is then transferred to form nicotinic acid adenine dinucleotide (NaAD). Finally, the nicotinic acid group in NaAD is amidated to an nicotinamide (Nam) group, forming nicotinamide adenine dinucleotide.^[1]

In a further step, some NAD⁺ is converted into NADP⁺ by NAD+ kinase, which phosphorylates NAD⁺.^[34] In most organisms, this enzyme uses ATP as the source of the phosphate group, although in bacteria such as Mycobacterium tuberculosis and in archaea such as Pyrococcus horikoshii, inorganic polyphosphate acts as an alternative phosphate donor.^[35][36]

Salvage pathways use three precursors for NAD+.

[edit] Salvage pathways

Besides assembling NAD⁺ de novo from simple amino acid precursors, cells also salvage preformed nicotinamide moieties. The three compounds that contain the nicotinamide ring and are used in these salvage metabolic pathways are nicotinic acid (Na), nicotinamide (Nam) and nicotinamide riboside (NR). These precursors are either taken up from the diet (in the form of vitamin B₃, also called niacin), or produced within cells when the nicotinamide moiety is released from NAD⁺ in ADP-ribose transfer reactions. They are fed into the NAD(P)⁺ biosynthetic pathway, shown above, through adenylation and phosphoribosylation reactions.^[1]

Despite the presence of the de novo pathway, these salvage reactions are essential in humans; a lack of niacin in the diet causes the vitamin deficiency disease pellagra.^[37] This high requirement for NAD⁺ is the result of the constant consumption of the coenzyme in signaling reactions, since the cycling of NAD⁺ between oxidized and reduced forms during redox reactions causes no change in the overall levels of the coenzyme.

Some pathogens, such as the yeast Candida glabrata and the bacterium Haemophilus influenzae are NAD⁺ auxotrophs. They are unable to synthesize the coenzyme and are therefore entirely dependent on nicotinamide salvage pathways.^[38][39]

[edit] History

The coenzyme NAD⁺ was first discovered by Arthur Harden and William Youndin 1906.^[40] They noticed that adding boiled and filtered yeast extract greatly accelerated alcoholic fermentation in unboiled yeast extracts. They called the unidentified factor responsible for this effect a coferment. Through a long and difficult purification from yeast extracts, this heat-stable factor was identified as a nucleotide sugar phosphate by Hans von Euler-Chelpin.^[41] In 1936, Otto Heinrich Warburg identified the function of the nucleotide in hydride transfer.^[42]

In the early 1940s, Arthur Kornberg made an important contribution towards understanding NAD(P) metabolism by being the first to isolate an enzyme from its biosynthetic pathway.^[43] Subsequently, in 1949, Morris Friedkin and Albert L. Lehninger proved that NADH linked metabolic pathways such as the citric acid cycle with the synthesis of ATP in oxidative phosphorylation.^[44]

[edit] See also

• Adenine
• Nucleotide
• Niacin

[edit] References

[edit] External links

• NAD and NADP
• Nicotinamide