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87-457: Nicotinamide adenine dinucleotide ( NAD ) 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 (H for hydrogen ), respectively. In cellular metabolism, NAD

174-641: A coenzyme in redox reactions, as a donor of ADP-ribose moieties in ADP-ribosylation reactions, as a precursor of the second messenger molecule cyclic ADP-ribose , as well as acting as a substrate for bacterial DNA ligases and a group of enzymes called sirtuins that use NAD to remove acetyl groups from proteins. In addition to these metabolic functions, NAD emerges as an adenine nucleotide that can be released from cells spontaneously and by regulated mechanisms, and can therefore have important extracellular roles. The main role of NAD in metabolism

261-447: A nucleotide , such as the electron carriers NAD and FAD , and coenzyme A , which carries acyl groups. Most of these cofactors are found in a huge variety of species, and some are universal to all forms of life. An exception to this wide distribution is a group of unique cofactors that evolved in methanogens , which are restricted to this group of archaea . Metabolism involves a vast array of chemical reactions, but most fall under

348-495: 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 ( de novo ) from either tryptophan or aspartic acid , each a case of an amino acid . Alternatively, more complex components of

435-513: A cofactor has been identified. Iodine is also an essential trace element, but this element is used as part of the structure of thyroid hormones rather than as an enzyme cofactor. Calcium is another special case, in that it is required as a component of the human diet, and it is needed for the full activity of many enzymes, such as nitric oxide synthase , protein phosphatases , and adenylate kinase , but calcium activates these enzymes in allosteric regulation , often binding to these enzymes in

522-419: 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 (Asp) in some bacteria and plants. The quinolinic acid is converted to nicotinic acid mononucleotide (NaMN) by transfer of a phosphoribose moiety. An adenylate moiety is then transferred to form nicotinic acid adenine dinucleotide (NaAD). Finally,

609-453: A complex with calmodulin . Calcium is, therefore, a cell signaling molecule, and not usually considered a cofactor of the enzymes it regulates. Other organisms require additional metals as enzyme cofactors, such as vanadium in the nitrogenase of the nitrogen-fixing bacteria of the genus Azotobacter , tungsten in the aldehyde ferredoxin oxidoreductase of the thermophilic archaean Pyrococcus furiosus , and even cadmium in

696-530: A different cofactor. This process of adapting a pre-evolved structure for a novel use is known as exaptation . Prebiotic origin of coenzymes . Like amino acids and nucleotides , certain vitamins and thus coenzymes can be created under early earth conditions. For instance, vitamin B3 can be synthesized with electric discharges applied to ethylene and ammonia . Similarly, pantetheine (a vitamin B5 derivative),

783-406: A few basic types of reactions that involve the transfer of functional groups . This common chemistry allows cells to use a small set of metabolic intermediates to carry chemical groups between different reactions. These group-transfer intermediates are the loosely bound organic cofactors, often called coenzymes . Each class of group-transfer reaction is carried out by a particular cofactor, which

870-553: A highly conserved structural motif, the idea that inhibitors based on NAD could be specific to one enzyme is surprising. However, this can be possible: for example, inhibitors based on the compounds mycophenolic acid and tiazofurin inhibit IMP dehydrogenase at the NAD binding site. Because of the importance of this enzyme in purine metabolism , these compounds may be useful as anti-cancer, anti-viral, or immunosuppressive drugs . Other drugs are not enzyme inhibitors, but instead activate enzymes involved in NAD metabolism. Sirtuins are

957-541: A long and difficult purification from yeast extracts, this heat-stable factor was identified as a nucleotide sugar phosphate by Hans von Euler-Chelpin . In 1936, the German scientist Otto Heinrich Warburg showed the function of the nucleotide coenzyme in hydride transfer and identified the nicotinamide portion as the site of redox reactions. Vitamin precursors of NAD were first identified in 1938, when Conrad Elvehjem showed that liver has an "anti-black tongue" activity in

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1044-435: A low-molecular-weight, non-protein organic compound that is loosely attached, participating in enzymatic reactions as a dissociable carrier of chemical groups or electrons; a prosthetic group is defined as a tightly bound, nonpolypeptide unit in a protein that is regenerated in each enzymatic turnover. Some enzymes or enzyme complexes require several cofactors. For example, the multienzyme complex pyruvate dehydrogenase at

1131-443: A molecular mass less than 1000 Da) that can be either loosely or tightly bound to the enzyme and directly participate in the reaction. In the latter case, when it is difficult to remove without denaturing the enzyme, it can be called a prosthetic group . There is no sharp division between loosely and tightly bound cofactors. Many such as NAD can be tightly bound in some enzymes, while it is loosely bound in others. Another example

1218-406: A part of the protein sequence. This often replaces the need for an external binding factor, such as a metal ion, for protein function. Potential modifications could be oxidation of aromatic residues, binding between residues, cleavage or ring-forming. These alterations are distinct from other post-translation protein modifications , such as phosphorylation , methylation , or glycosylation in that

1305-436: A particularly interesting target for such drugs, since activation of these NAD-dependent deacetylases extends lifespan in some animal models. Compounds such as resveratrol increase the activity of these enzymes, which may be important in their ability to delay aging in both vertebrate, and invertebrate model organisms . In one experiment, mice given NAD for one week had improved nuclear-mitochrondrial communication. Because of

1392-512: A precursor of coenzyme A and thioester-dependent synthesis, can be formed spontaneously under evaporative conditions. Other coenzymes may have existed early on Earth, such as pterins (a derivative of vitamin B9 ), flavins ( FAD , flavin mononucleotide = FMN), and riboflavin (vitamin B2). Changes in coenzymes . A computational method, IPRO, recently predicted mutations that experimentally switched

1479-423: A protein at some point, and then rebind later. Both prosthetic groups and cosubstrates have the same function, which is to facilitate the reaction of enzymes and proteins. An inactive enzyme without the cofactor is called an apoenzyme , while the complete enzyme with cofactor is called a holoenzyme . The International Union of Pure and Applied Chemistry (IUPAC) defines "coenzyme" a little differently, namely as

1566-427: A pyridine base. The three vitamin precursors used in these salvage metabolic pathways are nicotinic acid (NA), nicotinamide (Nam) and nicotinamide riboside (NR). These compounds can be taken up from the diet and are termed vitamin B 3 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

1653-559: A second peak in UV absorption at 339 nm with an extinction coefficient of 6,220 Mcm. 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

1740-601: A structural property. Different sources give slightly different definitions of coenzymes, cofactors, and prosthetic groups. Some consider tightly bound organic molecules as prosthetic groups and not as coenzymes, while others define all non-protein organic molecules needed for enzyme activity as coenzymes, and classify those that are tightly bound as coenzyme prosthetic groups. These terms are often used loosely. A 1980 letter in Trends in Biochemistry Sciences noted

1827-422: A subsequent reaction catalyzed by a different enzyme. In the latter case, the cofactor can also be considered a substrate or cosubstrate. Vitamins can serve as precursors to many organic cofactors (e.g., vitamins B 1 , B 2 , B 6 , B 12 , niacin , folic acid ) or as coenzymes themselves (e.g., vitamin C ). However, vitamins do have other functions in the body. Many organic cofactors also contain

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1914-434: 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 (nm), with an extinction coefficient of 16,900  M cm . NADH also absorbs at higher wavelengths, with

2001-418: Is thiamine pyrophosphate (TPP), which is tightly bound in transketolase or pyruvate decarboxylase , while it is less tightly bound in pyruvate dehydrogenase . Other coenzymes, flavin adenine dinucleotide (FAD), biotin , and lipoamide , for instance, are tightly bound. Tightly bound cofactors are, in general, regenerated during the same reaction cycle, while loosely bound cofactors can be regenerated in

2088-409: Is as a coenzyme in anabolic metabolism. In the name NAD, the superscripted plus sign indicates the positive formal charge on one of its nitrogen atoms. 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 (the 1' position) ( adenosine diphosphate ribose ) and

2175-422: Is as a precursor of cyclic ADP-ribose , which is produced from NAD by ADP-ribosyl cyclases, as part of a second messenger system . This molecule acts in calcium signaling by releasing calcium from intracellular stores. It does this by binding to and opening a class of calcium channels called ryanodine receptors , which are located in the membranes of organelles , such as the endoplasmic reticulum , and inducing

2262-474: 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

2349-563: Is conducted using X-ray crystallography and mass spectroscopy ; structural data is necessary because sequencing does not readily identify the altered sites. The term is used in other areas of biology to refer more broadly to non-protein (or even protein) molecules that either activate, inhibit, or are required for the protein to function. For example, ligands such as hormones that bind to and activate receptor proteins are termed cofactors or coactivators, whereas molecules that inhibit receptor proteins are termed corepressors. One such example

2436-413: 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

2523-406: 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

2610-516: Is in the release of energy from nutrients. Here, reduced compounds such as glucose and fatty acids are oxidized, thereby releasing energy. This energy is transferred to NAD by reduction to NADH, as part of beta oxidation , glycolysis , and the citric acid cycle . In eukaryotes the electrons carried by the NADH that is produced in the cytoplasm are transferred into the mitochondrion (to reduce mitochondrial NAD) by mitochondrial shuttles , such as

2697-425: 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

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2784-455: 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. In addition to these functions within the cell, a group of extracellular ADP-ribosyltransferases has recently been discovered, but their functions remain obscure. NAD may also be added onto cellular RNA as a 5'-terminal modification. Another function of this coenzyme in cell signaling

2871-401: Is often amplified in cancer cells. It has been studied for its potential use in the therapy of neurodegenerative diseases such as Alzheimer's and Parkinson's disease as well as multiple sclerosis . A placebo-controlled clinical trial of NADH (which excluded NADH precursors) in people with Parkinson's failed to show any effect. NAD is also a direct target of the drug isoniazid , which

2958-554: Is proposed to be a novel neurotransmitter that transmits information from nerves to effector cells in smooth muscle organs. In plants, the extracellular nicotinamide adenine dinucleotide induces resistance to pathogen infection and the first extracellular NAD receptor has been identified. Further studies are needed to determine the underlying mechanisms of its extracellular actions and their importance for human health and life processes in other organisms. The enzymes that make and use NAD and NADH are important in both pharmacology and

3045-535: Is still needed for anabolic reactions, these bacteria use a nitrite oxidoreductase to produce enough proton-motive force to run part of the electron transport chain in reverse, generating NADH. The coenzyme NAD is also consumed in ADP-ribose transfer reactions. For example, enzymes called ADP-ribosyltransferases add the ADP-ribose moiety of this molecule to proteins, in a posttranslational modification called ADP-ribosylation . ADP-ribosylation involves either

3132-516: 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 . Most organisms synthesize NAD from simple components. The specific set of reactions differs among organisms, but

3219-542: Is the substrate for a set of enzymes that produce it, and a set of enzymes that consume it. An example of this are the dehydrogenases that use nicotinamide adenine dinucleotide (NAD ) as a cofactor. Here, hundreds of separate types of enzymes remove electrons from their substrates and reduce NAD to NADH. This reduced cofactor is then a substrate for any of the reductases in the cell that require electrons to reduce their substrates. Therefore, these cofactors are continuously recycled as part of metabolism . As an example,

3306-601: Is the transfer of electrons from one molecule to another. Reactions of this type are catalyzed by a large group of enzymes called oxidoreductases . The correct names for these enzymes contain the names of both their substrates: for example NADH-ubiquinone oxidoreductase catalyzes the oxidation of NADH by coenzyme Q . However, these enzymes are also referred to as dehydrogenases or reductases , with NADH-ubiquinone oxidoreductase commonly being called NADH dehydrogenase or sometimes coenzyme Q reductase . There are many different superfamilies of enzymes that bind NAD / NADH. One of

3393-465: Is used as a supplement to culture media for some fastidious bacteria. The coenzyme NAD was first discovered by the British biochemists Arthur Harden and William John Young in 1906. 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

3480-513: Is used in the treatment of tuberculosis , an infection caused by Mycobacterium tuberculosis . Isoniazid is a prodrug and once it has entered the bacteria, it is activated by a peroxidase enzyme, which oxidizes the compound into a free radical form. This radical then reacts with NADH, to produce adducts that are very potent inhibitors of the enzymes enoyl-acyl carrier protein reductase , and dihydrofolate reductase . Since many oxidoreductases use NAD and NADH as substrates, and bind them using

3567-438: The aging process and to the pathogenesis of the chronic diseases of aging. Thus, the modulation of NAD may protect against cancer, radiation, and aging. In recent years, NAD has also been recognized as an extracellular signaling molecule involved in cell-to-cell communication. NAD is released from neurons in blood vessels , urinary bladder , large intestine , from neurosecretory cells, and from brain synaptosomes , and

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3654-559: The carbonic anhydrase from the marine diatom Thalassiosira weissflogii . In many cases, the cofactor includes both an inorganic and organic component. One diverse set of examples is the heme proteins, which consist of a porphyrin ring coordinated to iron . Iron–sulfur clusters are complexes of iron and sulfur atoms held within proteins by cysteinyl residues. They play both structural and functional roles, including electron transfer, redox sensing, and as structural modules. Organic cofactors are small organic molecules (typically

3741-426: 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

3828-564: The last universal ancestor , which lived about 4 billion years ago. Organic cofactors may have been present even earlier in the history of life on Earth. The nucleotide adenosine is a cofactor for many basic metabolic enzymes such as transferases. It may be a remnant of the RNA world . Adenosine-based cofactors may have acted as adaptors that allowed enzymes and ribozymes to bind new cofactors through small modifications in existing adenosine-binding domains , which had originally evolved to bind

3915-411: The malate-aspartate shuttle . The mitochondrial NADH is then oxidized in turn by the electron transport chain , which pumps protons across a membrane and generates ATP through oxidative phosphorylation . These shuttle systems also have the same transport function in chloroplasts . Since both the oxidized and reduced forms of nicotinamide adenine dinucleotide are used in these linked sets of reactions,

4002-618: The nucleotide adenosine monophosphate (AMP) as part of their structures, such as ATP , coenzyme A , FAD , and NAD . This common structure may reflect a common evolutionary origin as part of ribozymes in an ancient RNA world . It has been suggested that the AMP part of the molecule can be considered to be a kind of "handle" by which the enzyme can "grasp" the coenzyme to switch it between different catalytic centers. Cofactors can be divided into two major groups: organic cofactors , such as flavin or heme ; and inorganic cofactors , such as

4089-636: The DNA-AMP intermediate. Li et al. have found that NAD directly regulates protein-protein interactions. They also show that one of the causes of age-related decline in DNA repair may be increased binding of the protein DBC1 (Deleted in Breast Cancer 1) to PARP1 (poly[ADP–ribose] polymerase 1) as NAD levels decline during aging. The decline in cellular concentrations of NAD during aging likely contributes to

4176-539: The NADP/NADPH ratio is kept very low. Although it is important in catabolism, NADH is also used in anabolic reactions, such as gluconeogenesis . This need for NADH in anabolism poses a problem for prokaryotes growing on nutrients that release only a small amount of energy. For example, nitrifying bacteria such as Nitrobacter oxidize nitrite to nitrate, which releases sufficient energy to pump protons and generate ATP, but not enough to produce NADH directly. As NADH

4263-579: The acidic phosphate group of NADP. On the converse, in NAD-dependent enzymes the charge in this pocket is reversed, preventing NADP from binding. However, there are a few exceptions to this general rule, and enzymes such as aldose reductase , glucose-6-phosphate dehydrogenase , and methylenetetrahydrofolate reductase can use both coenzymes in some species. The redox reactions catalyzed by oxidoreductases are vital in all parts of metabolism, but one particularly important area where these reactions occur

4350-701: The activation of the transcription factor NAFC3 NAD is also consumed by different NAD+-consuming enzymes, such as CD38 , CD157 , PARPs and the NAD-dependent deacetylases ( sirtuins ,such as Sir2 .). 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. However, non-histone proteins can be deacetylated by sirtuins as well. These activities of sirtuins are particularly interesting because of their importance in

4437-466: The active site of an oxidoreductase, the nicotinamide ring of the coenzyme is positioned so that it can accept a hydride from the other substrate. Depending on the enzyme, the hydride donor is positioned either "above" or "below" the plane of the planar C4 carbon, as defined in the figure. Class A oxidoreductases transfer the atom from above; class B enzymes transfer it from below. Since the C4 carbon that accepts

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4524-462: The addition of a single ADP-ribose moiety, in mono-ADP-ribosylation , or the transferral of ADP-ribose to proteins in long branched chains, which is called poly(ADP-ribosyl)ation . Mono-ADP-ribosylation was first identified as the mechanism of a group of bacterial toxins , notably cholera toxin , but it is also involved in normal cell signaling . Poly(ADP-ribosyl)ation is carried out by the poly(ADP-ribose) polymerases . The poly(ADP-ribose) structure

4611-524: The amino acids typically acquire new functions. This increases the functionality of the protein; unmodified amino acids are typically limited to acid-base reactions, and the alteration of resides can give the protein electrophilic sites or the ability to stabilize free radicals. Examples of cofactor production include tryptophan tryptophylquinone (TTQ), derived from two tryptophan side chains, and 4-methylidene-imidazole-5-one (MIO), derived from an Ala-Ser-Gly motif. Characterization of protein-derived cofactors

4698-586: The author could not arrive at a single all-encompassing definition of a "coenzyme" and proposed that this term be dropped from use in the literature. Metal ions are common cofactors. The study of these cofactors falls under the area of bioinorganic chemistry . In nutrition , the list of essential trace elements reflects their role as cofactors. In humans this list commonly includes iron , magnesium , manganese , cobalt , copper , zinc , and molybdenum . Although chromium deficiency causes impaired glucose tolerance , no human enzyme that uses this metal as

4785-534: 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 . Nicotinamide adenine dinucleotide has several essential roles in metabolism . It acts as

4872-422: The cell maintains significant concentrations of both NAD and NADH, with the high NAD/NADH ratio allowing this coenzyme to act as both an oxidizing and a reducing agent. In contrast, the main function of NADPH is as a reducing agent in anabolism , with this coenzyme being involved in pathways such as fatty acid synthesis and photosynthesis . Since NADPH is needed to drive redox reactions as a strong reducing agent,

4959-411: The coenzymes are taken up from nutritive compounds such as niacin ; 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. Some NAD is converted into the coenzyme nicotinamide adenine dinucleotide phosphate (NADP), whose chemistry largely parallels that of NAD, though its predominant role

5046-550: The cofactor specificity of Candida boidinii xylose reductase from NADPH to NADH. Evolution of enzymes without coenzymes . If enzymes require a co-enzyme, how does the coenzyme evolve? The most likely scenario is that enzymes can function initially without their coenzymes and later recruit the coenzyme, even if the catalyzed reaction may not be as efficient or as fast. Examples are Alcohol Dehydrogenase (coenzyme: NAD⁺ ), Lactate Dehydrogenase (NAD⁺), Glutathione Reductase ( NADPH ). The first organic cofactor to be discovered

5133-415: The confusion in the literature and the essentially arbitrary distinction made between prosthetic groups and coenzymes group and proposed the following scheme. Here, cofactors were defined as an additional substance apart from protein and substrate that is required for enzyme activity and a prosthetic group as a substance that undergoes its whole catalytic cycle attached to a single enzyme molecule. However,

5220-420: The course of the day. This means that each ATP molecule is recycled 1000 to 1500 times daily. Organic cofactors, such as ATP and NADH , are present in all known forms of life and form a core part of metabolism . Such universal conservation indicates that these molecules evolved very early in the development of living things. At least some of the current set of cofactors may, therefore, have been present in

5307-449: The differences in the metabolic pathways of NAD biosynthesis between organisms, such as between bacteria and humans, this area of metabolism is a promising area for the development of new antibiotics . For example, the enzyme nicotinamidase , which converts nicotinamide to nicotinic acid, is a target for drug design, as this enzyme is absent in humans but present in yeast and bacteria. In bacteriology, NAD, sometimes referred to factor V,

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5394-432: The early 1960s. Studies in the 1980s and 1990s revealed the activities of NAD and NADP metabolites in cell signaling – such as the action of cyclic ADP-ribose , which was discovered in 1987. The metabolism of NAD remained an area of intense research into the 21st century, with interest heightened after the discovery of the NAD-dependent protein deacetylases called sirtuins in 2000, by Shin-ichiro Imai and coworkers in

5481-407: The early 20th century, with ATP being isolated in 1929 by Karl Lohmann, and coenzyme A being discovered in 1945 by Fritz Albert Lipmann . The functions of these molecules were at first mysterious, but, in 1936, Otto Heinrich Warburg identified the function of NAD in hydride transfer. This discovery was followed in the early 1940s by the work of Herman Kalckar , who established the link between

5568-532: The form of nicotinamide. Then, in 1939, he provided the first strong evidence that niacin is used to synthesize NAD. In the early 1940s, Arthur Kornberg was the first to detect an enzyme in the biosynthetic pathway. In 1949, the American biochemists 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. In 1958, Jack Preiss and Philip Handler discovered

5655-425: The hydrogen is prochiral , this can be exploited in enzyme kinetics to give information about the enzyme's mechanism. This is done by mixing an enzyme with a substrate that has deuterium atoms substituted for the hydrogens, so the enzyme will reduce NAD by transferring deuterium rather than hydrogen. In this case, an enzyme can produce one of two stereoisomers of NADH. Despite the similarity in how proteins bind

5742-544: The intermediates and enzymes involved in the biosynthesis of NAD; salvage synthesis from nicotinic acid is termed the Preiss-Handler pathway. In 2004, Charles Brenner and co-workers uncovered the nicotinamide riboside kinase pathway to NAD. The non-redox roles of NAD(P) were discovered later. The first to be identified was the use of NAD as the ADP-ribose donor in ADP-ribosylation reactions, observed in

5829-404: The junction of glycolysis and the citric acid cycle requires five organic cofactors and one metal ion: loosely bound thiamine pyrophosphate (TPP), covalently bound lipoamide and flavin adenine dinucleotide (FAD), cosubstrates nicotinamide adenine dinucleotide (NAD ) and coenzyme A (CoA), and a metal ion (Mg ). Organic cofactors are often vitamins or made from vitamins. Many contain

5916-520: The laboratory of Leonard P. Guarente . In 2009 Imai proposed the "NAD World" hypothesis that key regulators of aging and longevity in mammals are sirtuin 1 and the primary NAD synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT). In 2016 Imai expanded his hypothesis to "NAD World 2.0", which postulates that extracellular NAMPT from adipose tissue maintains NAD in the hypothalamus (the control center) in conjunction with myokines from skeletal muscle cells. In 2018, Napa Therapeutics

6003-399: The metal ions Mg , Cu , Mn and iron–sulfur clusters . Organic cofactors are sometimes further divided into coenzymes and prosthetic groups . The term coenzyme refers specifically to enzymes and, as such, to the functional properties of a protein. On the other hand, "prosthetic group" emphasizes the nature of the binding of a cofactor to a protein (tight or covalent) and, thus, refers to

6090-451: 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

6177-648: The most common superfamilies includes a structural motif known as the Rossmann fold . The motif is named after Michael Rossmann , who was the first scientist to notice how common this structure is within nucleotide-binding proteins. An example of a NAD-binding bacterial enzyme involved in amino acid metabolism that does not have the Rossmann fold is found in Pseudomonas syringae pv. tomato ( PDB : 2CWH ​; InterPro :  IPR003767 ). When bound in

6264-454: The nicotinamide absorbance of ~335 nm (near-UV), fluoresces at 445–460 nm (violet to blue) 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

6351-437: The nicotinamide ring. From the hydride electron pair, one electron is attracted to the slightly more electronegative atom of the nicotinamide ring of NAD, becoming part of the nicotinamide moiety. The second electron and proton atom are transferred to the carbon atom adjacent to 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

6438-579: The nicotinic acid moiety in NaAD is amidated to a nicotinamide (Nam) 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 (ATP) 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. Despite

6525-400: The other with nicotinamide at this position. The compound accepts or donates the equivalent of H. Such reactions (summarized in formula below) involve the removal of two hydrogen atoms from the reactant (R), in the form of a hydride ion (H), and a proton (H). The proton is released into solution, while the reductant RH 2 is oxidized and NAD reduced to NADH by transfer of the hydride to

6612-518: 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 (NAMPT), which produces nicotinamide mononucleotide (NMN). 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

6699-433: The oxidation of sugars and the generation of ATP. This confirmed the central role of ATP in energy transfer that had been proposed by Fritz Albert Lipmann in 1941. Later, in 1949, Morris Friedkin and Albert L. Lehninger proved that NAD linked metabolic pathways such as the citric acid cycle and the synthesis of ATP. In a number of enzymes, the moiety that acts as a cofactor is formed by post-translational modification of

6786-401: The presence of the de novo pathway, the salvage reactions are essential in humans; a lack of niacin 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

6873-471: 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. NAD

6960-463: 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(II). 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

7047-416: The regulation of aging . Other NAD-dependent enzymes include bacterial DNA ligases , which join two DNA ends by using NAD as a substrate to donate an adenosine monophosphate (AMP) moiety 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 . This contrasts with eukaryotic DNA ligases, which use ATP to form

7134-457: The research into future treatments for disease. Drug design and drug development exploits NAD in three ways: as a direct target of drugs, by designing enzyme inhibitors or activators based on its structure that change the activity of NAD-dependent enzymes, and by trying to inhibit NAD biosynthesis. Because cancer cells utilize increased glycolysis , and because NAD enhances glycolysis, nicotinamide phosphoribosyltransferase (NAD salvage pathway)

7221-431: The total quantity of ATP in the human body is about 0.1  mole . This ATP is constantly being broken down into ADP, and then converted back into ATP. Thus, at any given time, the total amount of ATP + ADP remains fairly constant. The energy used by human cells requires the hydrolysis of 100 to 150 moles of ATP daily, which is around 50 to 75 kg. In typical situations, humans use up their body weight of ATP over

7308-414: The two coenzymes, enzymes almost always show a high level of specificity for either NAD or NADP. This specificity reflects the distinct metabolic roles of the respective coenzymes, and is the result of distinct sets of amino acid residues in the two types of coenzyme-binding pocket. For instance, in the active site of NADP-dependent enzymes, an ionic bond is formed between a basic amino acid side-chain and

7395-416: The use of the term "cofactor" for inorganic substances; both types are included here. ) Coenzymes are further divided into two types. The first is called a " prosthetic group ", which consists of a coenzyme that is tightly (or even covalently) and permanently bound to a protein. The second type of coenzymes are called "cosubstrates", and are transiently bound to the protein. Cosubstrates may be released from

7482-496: Was NAD , which was identified by Arthur Harden and William Young 1906. 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 . Other cofactors were identified throughout

7569-424: Was formed to develop drugs against a novel aging-related target based on the research in NAD metabolism conducted in the lab of Eric Verdin . Cofactor (biochemistry) Cofactors can be classified into two types: inorganic ions and complex organic molecules called coenzymes . Coenzymes are mostly derived from vitamins and other organic essential nutrients in small amounts. (Some scientists limit

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