In glycolysis, what is the source of the electron that makes NAD+ into NADH instead of NADH+?

In glycolysis, what is the source of the electron that makes NAD+ into NADH instead of NADH+?

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I looked at the formula for the glycolysis reaction. The overall reaction seems balanced, however, I don't see anything on the left hand side of the equation that would provides the electron to convert NAD+ to NADH. Where does that electron come from?

Carbohydrate Metabolism for the MCAT: Everything You Need to Know

(Note: This guide is part of our MCAT Biochemistry series.)

Part 1: Introduction

Part 2: Digestion of carbohydrates

a) Enzymatic breakdown

b) Pancreatic regulation

c) Glycogenesis and glycogenolysis

Part 3: Glycolysis and fermentation

a) Glycolysis

b) Lactic acid fermentation

c) Gluconeogenesis

Part 4: Pyruvate oxidation and the TCA cycle

a) Mitochondrial structure

b) Pyruvate oxidation

c) The citric acid cycle

Part 5: Electron transport chain and oxidative phosphorylation

a) The electron transport chain

b) The electrochemical gradient

c) Oxidative phosphorylation

Part 6: Pentose phosphate pathway

Part 7: High-yield terms

Part 8: Passage-based questions and answers

Part 9: Standalone questions and answers

Control of Catabolic Pathways

Catabolic pathways are controlled by enzymes, proteins, electron carriers, and pumps that ensure that the remaining reactions can proceed.

Learning Objectives

Explain how catabolic pathways are controlled

Key Takeaways

Key Points

  • Glycolysis, the citric acid cycle, and the electron transport chain are catabolic pathways that bring forth non-reversible reactions.
  • Glycolysis control begins with hexokinase, which catalyzes the phosphorylation of glucose its product is glucose-6- phosphate, which accumulates when phosphofructokinase is inhibited.
  • The citric acid cycle is controlled through the enzymes that break down the reactions that make the first two molecules of NADH.
  • The rate of electron transport through the electron transport chain is affected by the levels of ADP and ATP, whereas specific enzymes of the electron transport chain are unaffected by feedback inhibition.

Key Terms

  • phosphofructokinase: any of a group of kinase enzymes that convert fructose phosphates to biphosphate
  • glycolysis: the cellular metabolic pathway of the simple sugar glucose to yield pyruvic acid and ATP as an energy source
  • kinase: any of a group of enzymes that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific target molecules (substrates) the process is termed phosphorylation

Control of Catabolic Pathways

Enzymes, proteins, electron carriers, and pumps that play roles in glycolysis, the citric acid cycle, and the electron transport chain tend to catalyze non-reversible reactions. In other words, if the initial reaction takes place, the pathway is committed to proceeding with the remaining reactions. Whether a particular enzyme activity is released depends upon the energy needs of the cell (as reflected by the levels of ATP, ADP, and AMP).


The control of glycolysis begins with the first enzyme in the pathway, hexokinase. This enzyme catalyzes the phosphorylation of glucose, which helps to prepare the compound for cleavage in a later step. The presence of the negatively-charged phosphate in the molecule also prevents the sugar from leaving the cell. When hexokinase is inhibited, glucose diffuses out of the cell and does not become a substrate for the respiration pathways in that tissue. The product of the hexokinase reaction is glucose-6-phosphate, which accumulates when a later enzyme, phosphofructokinase, is inhibited.

Glycolysis: The glycolysis pathway is primarily regulated at the three key enzymatic steps (1, 2, and 7) as indicated. Note that the first two steps that are regulated occur early in the pathway and involve hydrolysis of ATP.

Phosphofructokinase is the main enzyme controlled in glycolysis. High levels of ATP, citrate, or a lower, more acidic pH decrease the enzyme’s activity. An increase in citrate concentration can occur because of a blockage in the citric acid cycle. Fermentation, with its production of organic acids like lactic acid, frequently accounts for the increased acidity in a cell however, the products of fermentation do not typically accumulate in cells.

The last step in glycolysis is catalyzed by pyruvate kinase. The pyruvate produced can proceed to be catabolized or converted into the amino acid alanine. If no more energy is needed and alanine is in adequate supply, the enzyme is inhibited. The enzyme’s activity is increased when fructose-1,6-bisphosphate levels increase. (Recall that fructose-1,6-bisphosphate is an intermediate in the first half of glycolysis. ) The regulation of pyruvate kinase involves phosphorylation, resulting in a less-active enzyme. Dephosphorylation by a phosphatase reactivates it. Pyruvate kinase is also regulated by ATP (a negative allosteric effect).

If more energy is needed, more pyruvate will be converted into acetyl CoA through the action of pyruvate dehydrogenase. If either acetyl groups or NADH accumulate, there is less need for the reaction and the rate decreases. Pyruvate dehydrogenase is also regulated by phosphorylation: a kinase phosphorylates it to form an inactive enzyme, and a phosphatase reactivates it. The kinase and the phosphatase are also regulated.

Citric Acid Cycle

The citric acid cycle is controlled through the enzymes that catalyze the reactions that make the first two molecules of NADH. These enzymes are isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. When adequate ATP and NADH levels are available, the rates of these reactions decrease. When more ATP is needed, as reflected in rising ADP levels, the rate increases. α-Ketoglutarate dehydrogenase will also be affected by the levels of succinyl CoA, a subsequent intermediate in the cycle, causing a decrease in activity. A decrease in the rate of operation of the pathway at this point is not necessarily negative as the increased levels of the α-ketoglutarate not used by the citric acid cycle can be used by the cell for amino acid (glutamate) synthesis.

Citric Acid Cycle: Enzymes, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, catalyze the reactions that make the first two molecules of NADH in the citric acid cycle. Rates of the reaction decrease when sufficient ATP and NADH levels are reached.

Electron Transport Chain

Specific enzymes of the electron transport chain are unaffected by feedback inhibition, but the rate of electron transport through the pathway is affected by the levels of ADP and ATP. Greater ATP consumption by a cell is indicated by a buildup of ADP. As ATP usage decreases, the concentration of ADP decreases: ATP begins to build up in the cell. This change in the relative concentration of ADP to ATP triggers the cell to slow down the electron transport chain.

Electron Chain Transport: Levels of ADP and ATP affect the rate of electron transport through this type of chain transport.

Overview of Cellular Respiration

Figure 1. High-energy molecules: ATP and NADH. The top panel illustrates the hydrolysis of ATP to ADP. The standard free energy of this reaction is

7.3 kcal/mol. The lower panel illustrates the reduction of NAD+ to NADH + H+.

All cells require some source of energy to carry out their normal functions. The energy in cells is usually stored in the form of chemical bonds. In the next few tutorials you will learn about metabolic pathways (pathways of chemical reactions in a cell), including catabolic pathways, which describe reactions that breakdown molecules, and anabolic pathways, which describe reactions that build molecules. Often catabolic pathways release energy when chemical bonds are broken, whereas anabolic pathways may require energy to form chemical bonds. In plant cells, energy is derived from sunlight and used in anabolic pathways to synthesize simple sugars. These sugars can be stored and used later in either anabolic or catabolic pathways. In animal cells, energy is derived from the catabolism of ingested macromolecules such as starch and fat from other organisms (e.g. the hamburger you had for lunch). The large macromolecules are catabolized into simple sugars and other building blocks, releasing energy along the way. This energy is captured in the form of two types of high-energy molecules: ATP and electron carriers.

This tutorial describes the catabolism of glucose, the most common simple sugar found in both animals and plants. Remember from a previous tutorial (Properties of Macromolecules II: Nucleic Acids, Polysaccharides and Lipids), glucose is found in both glycogen and starch. The complete catabolism of glucose into CO2 and H2O is referred to as cellular respiration because it requires oxygen. The net reaction for cellular respiration is C6H12O6 + 6O2 -> 6CO2 + 6H2O + 38ATP. The catabolism of glucose occurs through a series of oxidation reactions. Recall from Biology 110 that the oxidationof a molecule involves the removal of electrons. The oxidation of organic molecules occurs by the removal of electrons and protons (H+). In biological reactions, an oxidation reaction is coupled to a reduction reaction (the addition of electrons and protons) such that one molecule is oxidized and the other is reduced. In the catabolism of glucose, sugars are oxidized in reactions that are coupled to the reduction of the most common electron carrier, nicotinamide adenine dinucleotide (NAD+), (Figure 1). For instance, in the following reaction: malate + NAD+ -> oxaloacetate + NADH + H+, malate is oxidized and NAD->is reduced. Cellular respiration occurs in a stepwise fashion, initially producing many molecules of reduced electron carriers (NADH and FADH2). These reduced electron carriers will eventually be oxidized in the mitochondria in a process that is linked to ATP synthesis. It is only in this final step that oxygen is actually used. The reduced electron carriers donate their electrons to an electron transport chain, and eventually, oxygen is reduced to yield water. This final step of cellular respiration yields the largest amount of energy, in the form of ATP.

There are four distinct stages of cellular respiration: glycolysis, the oxidation of glucose to the three-carbon sugar pyruvate pyruvate oxidation, the oxidation of pyruvate to acetyl coenzyme A (acetyl CoA) the citric acid cycle(also referred to as the Kreb's cycle or TCA cycle), the complete oxidation of acetyl CoA and finally, the oxidation of the reduced electron carriers linked to the synthesis of ATP. The first three stages (glycolysis, pyruvate oxidation and the citric acid cycle) will be described in this tutorial. In addition, we will consider the process of fermentation, which occurs in the absence of oxygen, whereby pyruvate is reduced and a variety of by-products are generated. The final step of cellular respiration, the oxidation of the electron carriers linked to ATP synthesis, will be covered in the next tutorial.


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 the other with nicotinamide at this position. [1] [2]

The compound accepts or donates the equivalent of H − . [3] 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 RH2 is oxidized and NAD + reduced to NADH by transfer of the hydride to the nicotinamide ring.

From the hydride electron pair, one electron is transferred to the positively charged nitrogen of the nicotinamide ring of NAD + , and the second hydrogen atom transferred to the C4 carbon atom opposite this nitrogen. The midpoint potential of the NAD + /NADH redox pair is −0.32 volts, which makes NADH a strong reducing agent. [4] 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. [2]

In appearance, all forms of this coenzyme are white amorphous powders that are hygroscopic and highly water-soluble. [5] 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 acids or alkalis. Upon decomposition, they form products that are enzyme inhibitors. [6]

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 −1 cm −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 −1 cm −1 . [7] 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. [7]

NAD + and NADH also differ in their fluorescence. Freely diffusing NADH in aqueous solution, when excited at 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. [8] [9] 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. [9] [10] These changes in fluorescence are also used to measure changes in the redox state of living cells, through fluorescence microscopy. [11]

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. [12] The actual concentration of NAD + in cell cytosol is harder to measure, with recent estimates in animal cells ranging around 0.3 mM, [13] [14] and approximately 1.0 to 2.0 mM in yeast. [15] However, more than 80% of NADH fluorescence in mitochondria is from bound form, so the concentration in solution is much lower. [16]

NAD + concentrations are highest in the mitochondria, constituting 40% to 70% of the total cellular NAD + . [17] NAD + in the cytosol is carried into the mitochondrion by a specific membrane transport protein, since the coenzyme cannot diffuse across membranes. [18] The intracellular half-life of NAD + was claimed to be between 1–2 hours by one review, [19] whereas another review gave varying estimates based on compartment: intracellular 1–4 hours, cytoplasmic 2 hours, and mitochondrial 4–6 hours. [20]

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. [21] 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 between free NAD + and NADH in the cytoplasm typically lie around 700:1 the ratio is thus favourable for oxidative reactions. [22] [23] The ratio of total NAD + /NADH is much lower, with estimates ranging from 3–10 in mammals. [24] In contrast, the NADP + /NADPH ratio is normally about 0.005, so NADPH is the dominant form of this coenzyme. [25] These different ratios are key to the different metabolic roles of NADH and NADPH.

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. [26]

De novo production Edit

Most organisms synthesize NAD + from simple components. [3] The specific set of reactions differs among organisms, 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 (Asp) in some bacteria and plants. [27] [28] 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, the nicotinic acid moiety in NaAD is amidated to a nicotinamide (Nam) moiety, forming nicotinamide adenine dinucleotide. [3]

In a further step, some NAD + is converted into NADP + by NAD + kinase, which phosphorylates NAD + . [29] In most organisms, this enzyme uses 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. [30] [31]

Salvage pathways Edit

Despite 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. [32] 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. [3] The major source of NAD + in mammals is the salvage pathway which recycles the nicotinamide produced by enzymes utilizing NAD + . [33] The first step, and the rate-limiting enzyme in the salvage pathway is nicotinamide phosphoribosyltransferase (NAMPT), which produces nicotinamide mononucleotide (NMN). [33] NMN is the immediate precursor to NAD+ in the salvage pathway. [34]

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 (NA), nicotinamide (Nam) and nicotinamide riboside (NR). [3] These compounds can be taken up from the diet and are termed vitamin B3 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. [35] There are some reports that mammalian cells can take up extracellular NAD + from their surroundings, [36] and both nicotinamide and nicotinamide riboside can be absorbed from the gut. [37]

The salvage pathways used in microorganisms differ from those of mammals. [38] 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. [39] [40] 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. [41]

Nicotinamide adenine dinucleotide has several essential roles in metabolism. It acts as 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, [43] [44] and can therefore have important extracellular roles. [44]

Oxidoreductase binding of NAD Edit

The main role of NAD + in metabolism 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. [45] 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. [46]

There are many different superfamilies of enzymes that bind NAD + / NADH. One of the most common superfamilies include a structural motif known as the Rossmann fold. [47] [48] The motif is named after Michael Rossmann who was the first scientist to notice how common this structure is within nucleotide-binding proteins. [49]

An example of a NAD-binding bacterial enzyme involved in amino acid metabolism that does not have Rossmann fold is found in Pseudomonas syringae pv. tomato ( PDB: 2CWH ​ InterPro: IPR003767). [50]

When bound in 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 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. [51]

Despite the similarity in how proteins bind the two coenzymes, enzymes almost always show a high level of specificity for either NAD + or NADP + . [52] 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 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. [53]

Role in redox metabolism Edit

The redox reactions catalyzed by oxidoreductases are vital in all parts of metabolism, but one particularly important function of these reactions is to enable nutrients to unlock the energy stored in the relatively weak double bond of oxygen. [54] Here, reduced compounds such as glucose and fatty acids are oxidized, thereby releasing the chemical energy of O2. In this process, NAD + is reduced 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 the malate-aspartate shuttle. [55] 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. [56] These shuttle systems also have the same transport function in chloroplasts. [57]

Since both the oxidized and reduced forms of nicotinamide adenine dinucleotide are used in these linked sets of reactions, 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. [58] 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, the NADP + /NADPH ratio is kept very low. [58]

Although it is important in catabolism, NADH is also used in anabolic reactions, such as gluconeogenesis. [59] 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. [60] As NADH 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. [61]

Non-redox roles Edit

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. [62] ADP-ribosylation involves either 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. [63] 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. [64] [65] Poly(ADP-ribosyl)ation is carried out by the poly(ADP-ribose) polymerases. [63] [66] 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. [66] In addition to these functions within the cell, a group of extracellular ADP-ribosyltransferases has recently been discovered, but their functions remain obscure. [67] NAD + may also be added onto cellular RNA as a 5'-terminal modification. [68]

Another function of this coenzyme in cell signaling is as a precursor of cyclic ADP-ribose, which is produced from NAD + by ADP-ribosyl cyclases, as part of a second messenger system. [69] This molecule acts in calcium signaling by releasing calcium from intracellular stores. [70] 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. [71]

NAD + is also consumed by sirtuins, which are NAD-dependent deacetylases, such as Sir2. [72] 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. [73] However, non-histone proteins can be deacetylated by sirtuins as well. These activities of sirtuins are particularly interesting because of their importance in the regulation of aging. [74]

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. [75] This contrasts with eukaryotic DNA ligases, which use ATP to form the DNA-AMP intermediate. [76]

Li et al. have found that NAD + directly regulates protein-protein interactions. [77] 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. [77] Thus, the modulation of NAD + may protect against cancer, radiation, and aging. [77]

Extracellular actions of NAD + Edit

In recent years, NAD + has also been recognized as an extracellular signaling molecule involved in cell-to-cell communication. [44] [78] [79] NAD + is released from neurons in blood vessels, [43] urinary bladder, [43] [80] large intestine, [81] [82] from neurosecretory cells, [83] and from brain synaptosomes, [84] and is proposed to be a novel neurotransmitter that transmits information from nerves to effector cells in smooth muscle organs. [81] [82] In plants, the extracellular nicotinamide adenine dinucleotide induces resistance to pathogen infection and the first extracellular NAD receptor has been identified. [85] 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 the research into future treatments for disease. [86] 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. [87]

Because cancer cells utilize increased glycolysis, and because NAD enhances glycolysis, nicotinamide phosphoribosyltransferase (NAD salvage pathway) is often amplified in cancer cells. [88] [89]

It has been studied for its potential use in the therapy of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. [3] A placebo-controlled clinical trial of NADH (which excluded NADH precursors) in people with Parkinson's failed to show any effect. [90]

NAD + is also a direct target of the drug isoniazid, which 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. [91] This radical then reacts with NADH, to produce adducts that are very potent inhibitors of the enzymes enoyl-acyl carrier protein reductase, [92] and dihydrofolate reductase. [93]

Since a large number of oxidoreductases use NAD + and NADH as substrates, and bind them using a highly conserved structural motif, the idea that inhibitors based on NAD + could be specific to one enzyme is surprising. [94] 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. [94] [95] Other drugs are not enzyme inhibitors, but instead activate enzymes involved in NAD + metabolism. Sirtuins are a particularly interesting target for such drugs, since activation of these NAD-dependent deacetylases extends lifespan in some animal models. [96] Compounds such as resveratrol increase the activity of these enzymes, which may be important in their ability to delay aging in both vertebrate, [97] and invertebrate model organisms. [98] [99] In one experiment, mice given NAD for one week had improved nuclear-mitochrondrial communication. [100]

Because of 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. [101] [102] 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. [38]

In bacteriology, NAD, sometimes referred to factor V, is used a supplement to culture media for some fastidious bacteria. [103]

The coenzyme NAD + was first discovered by the British biochemists Arthur Harden and William John Young in 1906. [104] 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. [105] 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. [106]

Vitamin precursors of NAD + were first identified in 1938, when Conrad Elvehjem showed that liver has an "anti-black tongue" activity in the form of nicotinamide. [107] Then, in 1939, he provided the first strong evidence that niacin is used to synthesize NAD + . [108] In the early 1940s, Arthur Kornberg was the first to detect an enzyme in the biosynthetic pathway. [109] 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. [110] In 1958, Jack Preiss and Philip Handler discovered the intermediates and enzymes involved in the biosynthesis of NAD + [111] [112] 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 + . [113]

The non-redox roles of NAD(P) were discovered later. [2] The first to be identified was the use of NAD + as the ADP-ribose donor in ADP-ribosylation reactions, observed in the early 1960s. [114] 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. [115]

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 the laboratory of Leonard P. Guarente. [116] 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). [117] 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. [118]

Fermentation and Regeneration of NAD+

Any discussion that focuses on fermentation should dwell on fermentation of pyruvate. However, some of the core principles of fermentation are visible in many examples in day-to-day activities. It does not matter how small a molecule is fermentation and regeneration of NAD+ is possible.

The Role of Fermentation

Oxidation of small organic compounds takes place through microorganism that gets their energy from cellular maintenance and growth. An example is the oxidation of glucose through glycoses.

Some essential steps needed for glucose to ferment involve the reduction of an electron NAD+ to NADH. During glycosis, cells will generate large amounts of NADH and deplete all the NAD+ supply. For glycosis to continue, the cell must find a way to regenerate NAD+ either through synthesis or recycling.

If there is no other option or process to take place, no one can tell what the cell might do. We can try putting back the electrons that were earlier stripped off the glucose into the downstream product or one of its derivatives. Fermentation is when we try to restore pools of oxidizing agents (the earlier removed electron).

An Example of Fermentation: Lactic Acid

This is an everyday example where the reduction of the compound to lactate by the lactic acid takes place through fermentation.

This reaction is what happens to your muscles during exercises. During the exercise, your muscles require large amounts of Adenosine Triphosphate (ATP) to perform the selected activity. Once the ATP go down, the muscle fibers will not keep up with the increasing demand for respiration because oxygen levels are becoming limited and Nicotinamide adenine dinucleotide (NADH) accumulates. The cells need to get rid of the excess and regenerate NAD+, and so the pyruvate will assume the role of an electron acceptor and start generating lactate and oxidizing NADH to NAD+. Most bacteria will use this pathway for the NADH /NAD+ cycle to complete. This is exactly what happens in yogurt.

Where is The Energy Coming From in Fermentation?

The reacting agents, in this case, are the Proton, NADH, and the Pyruvate. The products are NAD+ and lactate. The entire fermentation process gives reduced pyruvate by forming lactic acid the oxidation of NADH to form NAD+. The electrons from NADH and the proton combine to reduce pyruvate into lactate. If we examine this reaction, we will see that in normal conditions, the transfer of electrons from NADH to pyruvate to form lactate is an exogenic reaction and therefore a thermodynamic outcome. The reduction and the oxidation phases of the fermentation process are linked and catalyzed by the enzyme lactate dehydrogenase.

Nature has Several Fermentation Pathways

Nature as we know it has evolved to complete the NADH / NAD+ cycle. It is important that we understand the general concepts of fermentation. Generally, cells try to maintain a balance or a constant ratio between NADH and NAD+ when the ratio becomes unstable the cells try to compensate by modulating their cellular activities. The only requirement that makes fermentation a possibility is the use of a small compound (organic) as an electron acceptor for NADH and regenerates to NAD+. Read more about natural sources of NAD+.

Fermentation Without Substrate-Level Phosphorylation

Fermentation is the process of extracting energy from the oxidation of organic compounds such as carbohydrates.

Learning Objectives

Give examples of various types of fermentation: homolactic, heterolactic and alcoholic

Key Takeaways

Key Points

  • Fermentation without substrate level phosphorylation uses an endogenous electron acceptor, which is usually an organic compound.
  • Fermentation is important in anaerobic conditions when there is no oxidative phosphorylation to maintain the production of ATP (adenosine triphosphate) by glycolysis.
  • During fermentation, pyruvate is metabolised to various compounds such as lactic acid, ethanol and carbon dioxide or other acids.

Key Terms

  • fermentation: Any of many anaerobic biochemical reactions in which an enzyme (or several enzymes produced by a microorganism) catalyses the conversion of one substance into another especially the conversion (using yeast) of sugars to alcohol or acetic acid with the evolution of carbon dioxide.
  • substrate: a surface on which an organism grows or to which it is attached
  • oxidative phosphorylation: Oxidative phosphorylation (or OXPHOS in short) is a metabolic pathway that uses energy released by the oxidation of nutrients to produce adenosine triphosphate (ATP).
  • electron acceptor: An electron acceptor is a chemical entity that accepts electrons transferred to it from another compound. It is an oxidizing agent that, by virtue of its accepting electrons, is itself reduced in the process.

Pyruvic acid: Pyruvic acid can be made from glucose through glycolysis, converted back to carbohydrates (such as glucose) via gluconeogenesis, or to fatty acids through acetyl-CoA. It can also be used to construct the amino acid alanine and be converted into ethanol. Pyruvic acid supplies energy to living cells through the citric acid cycle (also known as the Krebs cycle) when oxygen is present (aerobic respiration), and alternatively ferments to produce lactic acid when oxygen is lacking (fermentation).

Fermentation is the process of extracting energy from the oxidation of organic compounds, such as carbohydrates, using an endogenous electron acceptor, which is usually an organic compound. In contrast, respiration is where electrons are donated to an exogenous electron acceptor, such as oxygen, via an electron transport chain. Fermentation is important in anaerobic conditions when there is no oxidative phosphorylation to maintain the production of ATP (adenosine triphosphate) by glycolysis.

During fermentation, pyruvate is metabolised to various compounds. Homolactic fermentation is the production of lactic acid from pyruvate alcoholic fermentation is the conversion of pyruvate into ethanol and carbon dioxide and heterolactic fermentation is the production of lactic acid as well as other acids and alcohols. Fermentation does not necessarily have to be carried out in an anaerobic environment. For example, even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to oxidative phosphorylation, as long as sugars are readily available for consumption (a phenomenon known as the Crabtree effect). The antibiotic activity of Hops also inhibits aerobic metabolism in Yeast.

Sugars are the most common substrate of fermentation, and typical examples of fermentation products are ethanol, lactic acid, lactose, and hydrogen. However, more exotic compounds can be produced by fermentation, such as butyric acid and acetone. Yeast carries out fermentation in the production of ethanol in beers, wines, and other alcoholic drinks, along with the production of large quantities of carbon dioxide. Fermentation occurs in mammalian muscle during periods of intense exercise where oxygen supply becomes limited, resulting in the creation of lactic acid.

First Half of Glycolysis (Energy-Requiring Steps)

Figure 2. The first half of glycolysis uses two ATP molecules in the phosphorylation of glucose, which is then split into two three-carbon molecules.

Step 1. The first step in glycolysis is catalyzed by hexokinase, an enzyme with broad specificity that catalyzes the phosphorylation of six-carbon sugars. Hexokinase phosphorylates glucose using ATP as the source of the phosphate, producing glucose-6-phosphate, a more reactive form of glucose. This reaction prevents the phosphorylated glucose molecule from continuing to interact with the GLUT proteins, and it can no longer leave the cell because the negatively charged phosphate will not allow it to cross the hydrophobic interior of the plasma membrane.

Step 2. In the second step of glycolysis, an isomerase converts glucose-6-phosphate into one of its isomers, fructose-6-phosphate. An isomerase is an enzyme that catalyzes the conversion of a molecule into one of its isomers. This change from phosphoglucose to phosphofructose allows the eventual split of the sugar into two three-carbon molecules.

Step 3. The third step is the phosphorylation of fructose-6-phosphate, catalyzed by the enzyme phosphofructokinase. A second ATP molecule donates a high-energy phosphate to fructose-6-phosphate, producing fructose-1,6-bisphosphate. In this pathway, phosphofructokinase is a rate-limiting enzyme. It is active when the concentration of ADP is high it is less active when ADP levels are low and the concentration of ATP is high. Thus, if there is “sufficient” ATP in the system, the pathway slows down. This is a type of end product inhibition, since ATP is the end product of glucose catabolism.

Step 4. The newly added high-energy phosphates further destabilize fructose-1,6-bisphosphate. The fourth step in glycolysis employs an enzyme, aldolase, to cleave 1,6-bisphosphate into two three-carbon isomers: dihydroxyacetone-phosphate and glyceraldehyde-3-phosphate.

Step 5. In the fifth step, an isomerase transforms the dihydroxyacetone-phosphate into its isomer, glyceraldehyde-3-phosphate. Thus, the pathway will continue with two molecules of a single isomer. At this point in the pathway, there is a net investment of energy from two ATP molecules in the breakdown of one glucose molecule.

How many ATP are produced from each NADH that enters the electron transport chain?

is NADH 2.5 or 3 ATP? To pass the electrons from NADH to last Oxygen acceptor,total of 10 protons are transported from matrix to inter mitochondrial membrane. 4 protons via complex 1,4 via complex 3 and 2 via complex 4. Thus for NADH&mdash 10/4=2.5 ATP is produced actually. Similarly for 1 FADH2, 6 protons are moved so 6/4= 1.5 ATP is produced.

Consequently, how many ATP can be made from each NADH during the electron transport process?

Electron transport begins with several molecules of NADH and FADH2 from the Krebs cycle and transfers their energy into as many as 34 more ATP molecules. All told, then, up to 38 molecules of ATP can be produced from just one molecule of glucose in the process of aerobic respiration.

Cellular respiration produces 36 total ATP per molecule of glucose across three stages. Breaking the bonds between carbons in the glucose molecule releases energy. There are also high energy electrons captured in the form of 2 NADH (electron carriers) which will be utilized later in the electron transport chain.

Summary – NAD+ vs NADH vs NADPH

NAD + NADH and NADPH are coenzymes which participate in biological reactions. They are derivatives of vitamin B3 or niacin. They participate in redox reactions. In summary of the difference between NAD + NADH and NADPH, the NAD + is in the oxidized form of NADH while NADH is the reduced form of NAD + . NADPH, on the other hand, consists of an additional phosphate group than NADH and generates through the pentose phosphate pathway. Furthermore, NAD + and NADH participate in catabolic reactions while NADPH involves in anabolic reactions. Also, NAD + is an oxidizing agent while NADH and NADPH are reducing agents.


1. Ying, W. “NAD /NADH and NADP /NADPH in Cellular Functions and Cell Death: Regulation and Biological Consequences.” Current Neurology and Neuroscience Reports., U.S. National Library of Medicine, Feb. 2008. Available here

Image Courtesy:

1.”NAD+ Oxidation and Reduction”By JacobShalk – Own work, (CC BY-SA 4.0) via Commons Wikimedia
2.”NADH phys”By NEUROtiker – Own work, (Public Domain) via Commons Wikimedia
3.”NADPH”By Tagm2A at French Wikipedia – Own work (Public Domain) via Commons Wikimedia