9.3: Fermentation and Regeneration of NAD+ - Biology

Fermentation and regeneration of NAD+

Section summary

This section discusses the process of fermentation. Due to the heavy emphasis in this course on central carbon metabolism, the discussion of fermentation understandably focuses on the fermentation of pyruvate. Nevertheless, some of the core principles that we cover in this section apply equally well to the fermentation of many other small molecules.

The "purpose" of fermentation

The oxidation of a variety of small organic compounds is a process that is utilized by many organisms to garner energy for cellular maintenance and growth. The oxidation of glucose via glycolysis is one such pathway. Several key steps in the oxidation of glucose to pyruvate involve the reduction of the electron/energy shuttle NAD+ to NADH. You were already asked to figure out what options the cell might reasonably have to reoxidize the NADH to NAD+ in order to avoid consuming the available pools of NAD+ and to thus avoid stopping glycolysis. Put differently, during glycolysis, cells can generate large amounts of NADH and slowly exhaust their supplies of NAD+. If glycolysis is to continue, the cell must find a way to regenerate NAD+, either by synthesis or by some form of recycling.

In the absence of any other process—that is, if we consider glycolysis alone—it is not immediately obvious what the cell might do. One choice is to try putting the electrons that were once stripped off of the glucose derivatives right back onto the downstream product, pyruvate, or one of its derivatives. We can generalize the process by describing it as the returning of electrons to the molecule that they were once removed, usually to restore pools of an oxidizing agent. This, in short, is fermentation. As we will discuss in a different section, the process of respiration can also regenerate the pools of NAD+ from NADH. Cells lacking respiratory chains or in conditions where using the respiratory chain is unfavorable may choose fermentation as an alternative mechanism for garnering energy from small molecules.

An example: lactic acid fermentation

An everyday example of a fermentation reaction is the reduction of pyruvate to lactate by the lactic acid fermentation reaction. This reaction should be familiar to you: it occurs in our muscles when we exert ourselves during exercise. When we exert ourselves, our muscles require large amounts of ATP to perform the work we are demanding of them. As the ATP is consumed, the muscle cells are unable to keep up with the demand for respiration, O2 becomes limiting, and NADH accumulates. Cells need to get rid of the excess and regenerate NAD+, so pyruvate serves as an electron acceptor, generating lactate and oxidizing NADH to NAD+. Many bacteria use this pathway as a way to complete the NADH/NAD+ cycle. You may be familiar with this process from products like sauerkraut and yogurt. The chemical reaction of lactic acid fermentation is the following:

Pyruvate + NADH ↔ lactic acid + NAD+

Figure 1. Lactic acid fermentation converts pyruvate (a slightly oxidized carbon compound) to lactic acid. In the process, NADH is oxidized to form NAD+. Attribution: Marc T. Facciotti (original work)

Energy story for the fermentation of pyruvate to lactate

An example (if a bit lengthy) energy story for lactic acid fermentation is the following:

The reactants are pyruvate, NADH, and a proton. The products are lactate and NAD+. The process of fermentation results in the reduction of pyruvate to form lactic acid and the oxidation of NADH to form NAD+. Electrons from NADH and a proton are used to reduce pyruvate into lactate. If we examine a table of standard reduction potential, we see under standard conditions that a transfer of electrons from NADH to pyruvate to form lactate is exergonic and thus thermodynamically spontaneous. The reduction and oxidation steps of the reaction are coupled and catalyzed by the enzyme lactate dehydrogenase.

A second example: alcohol fermentation

Another familiar fermentation process is alcohol fermentation, which produces ethanol, an alcohol. The alcohol fermentation reaction is the following:

Figure 2. Ethanol fermentation is a two-step process. Pyruvate (pyruvic acid) is first converted into carbon dioxide and acetaldehyde. The second step converts acetaldehyde to ethanol and oxidizes NADH to NAD+. Facciotti (original work)

In the first reaction, a carboxyl group is removed from pyruvic acid, releasing carbon dioxide as a gas (some of you may be familiar with this as a key component of various beverages). The second reaction removes electrons from NADH, forming NAD+ and producing ethanol (another familiar compound—usually in the same beverage) from the acetaldehyde, which accepts the electrons.

Suggested discussion

Write a complete energy story for alcohol fermentation. Propose possible benefits of this type of fermentation for the single-celled yeast organism.

Fermentation pathways are numerous

While the lactic acid fermentation and alcohol fermentation pathways described above are examples, there are many more reactions (too numerous to go over) that Nature has evolved to complete the NADH/NAD+ cycle. It is important that you understand the general concepts behind these reactions. In general, cells try to maintain a balance or constant ratio between NADH and NAD+; when this ratio becomes unbalanced, the cell compensates by modulating other reactions to compensate. The only requirement for a fermentation reaction is that it uses a small organic compound as an electron acceptor for NADH and regenerates NAD+. Other familiar fermentation reactions include ethanol fermentation (as in beer and bread), propionic fermentation (it's what makes the holes in Swiss cheese), and malolactic fermentation (it's what gives Chardonnay its more mellow flavor—the more conversion of malate to lactate, the softer the wine). In Figure 3, you can see a large variety of fermentation reactions that various bacteria use to reoxidize NADH to NAD+. All of these reactions start with pyruvate or a derivative of pyruvate metabolism, such as oxaloacetate or formate. Pyruvate is produced from the oxidation of sugars (glucose or ribose) or other small, reduced organic molecules. It should also be noted that other compounds can be used as fermentation substrates besides pyruvate and its derivatives. These include methane fermentation, sulfide fermentation, or the fermentation of nitrogenous compounds such as amino acids. You are not expected to memorize all of these pathways. You are, however, expected to recognize a pathway that returns electrons to products of the compounds that were originally oxidized to recycle the NAD+/NADH pool and to associate that process with fermentation.

Figure 3. This figure shows various fermentation pathways using pyruvate as the initial substrate. In the figure, pyruvate is reduced to a variety of products via different and sometimes multistep (dashed arrows represent possible multistep processes) reactions. All details are deliberately not shown. The key point is to appreciate that fermentation is a broad term not solely associated with the conversion of pyruvate to lactic acid or ethanol. Source: Marc T. Facciotti (original work)

A note on the link between substrate-level phosphorylation and fermentation

Fermentation occurs in the absence of molecular oxygen (O2). It is an anaerobic process. Notice there is no O2 in any of the fermentation reactions shown above. Many of these reactions are quite ancient, hypothesized to be some of the first energy-generating metabolic reactions to evolve. This makes sense if we consider the following:

  1. The early atmosphere was highly reduced, with little molecular oxygen readily available.
  2. Small, highly reduced organic molecules were relatively available, arising from a variety of chemical reactions.
  3. These types of reactions, pathways, and enzymes are found in many different types of organisms, including bacteria, archaea, and eukaryotes, suggesting these are very ancient reactions.
  4. The process evolved long before O2 was found in the environment.
  5. The substrates, highly reduced, small organic molecules, like glucose, were readily available.
  6. The end products of many fermentation reactions are small organic acids, produced by the oxidation of the initial substrate.
  7. The process is coupled to substrate-level phosphorylation reactions. That is, small, reduced organic molecules are oxidized, and ATP is generated by first a red/ox reaction followed by the substrate-level phosphorylation.
  8. This suggests that substrate-level phosphorylation and fermentation reactions coevolved.

Suggested discussion

If the hypothesis is correct that substrate-level phosphorylation and fermentation reactions co-evolved and were the first forms of energy metabolism that cells used to generate ATP, then what would be the consequences of such reactions over time? What if these were the only forms of energy metabolism available over hundreds of thousands of years? What if cells were isolated in a small, closed environment? What if the small, reduced substrates were not being produced at the same rate of consumption during this time?

Consequences of fermentation

Imagine a world where fermentation is the primary mode for extracting energy from small molecules. As populations thrive, they reproduce and consume the abundance of small, reduced organic molecules in the environment, producing acids. One consequence is the acidification (decrease in pH) of the environment, including the internal cellular environment. This can be disruptive, since changes in pH can have a profound influence on the function and interactions among various biomolecules. Therefore, mechanisms needed to evolve that could remove the various acids. Fortunately, in an environment rich in reduced compounds, substrate-level phosphorylation and fermentation can produce large quantities of ATP.

It is hypothesized that this scenario was the beginning of the evolution of the F0F1-ATPase, a molecular machine that hydrolyzes ATP and translocates protons across the membrane (we'll see this again in the next section). With the F0F1-ATPase, the ATP produced from fermentation could now allow for the cell to maintain pH homeostasis by coupling the free energy of hydrolysis of ATP to the transport of protons out of the cell. The downside is that cells are now pumping all of these protons into the environment, which will now start to acidify.

Suggested discussion

If the hypothesis is correct that the F0F1-ATPase also co-evolved with substrate-level phosphorylation and fermentation reactions, then what would happen over time to the environment? While small, reduced organic compounds may have been initially abundant, if fermentation "took off" at some point, then the reduced compounds would run out and ATP might then become scarce as well. That's a problem. Thinking with the design challenge rubric in mind, define the problem(s) facing the cell in this hypothesized environment. What are other potential mechanisms or ways Nature could overcome the problem(s)?

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+.

Section Summary

If NADH cannot be metabolized through aerobic respiration, another electron acceptor is used. Most organisms will use some form of fermentation to accomplish the regeneration of NAD + , ensuring the continuation of glycolysis. The regeneration of NAD + in fermentation is not accompanied by ATP production therefore, the potential for NADH to produce ATP using an electron transport chain is not utilized.

Additional Self Check Questions

1. Tremetol, a metabolic poison found in white snake root plant, prevents the metabolism of lactate. When cows eat this plant, Tremetol is concentrated in the milk. Humans who consume the milk become ill. Symptoms of this disease, which include vomiting, abdominal pain, and tremors, become worse after exercise. Why do you think this is the case?

2. When muscle cells run out of oxygen, what happens to the potential for energy extraction from sugars and what pathways do the cell use?



anaerobic cellular respiration: the use of an electron acceptor other than oxygen to complete metabolism using electron transport-based chemiosmosis

fermentation: the steps that follow the partial oxidation of glucose via glycolysis to regenerate NAD + occurs in the absence of oxygen and uses an organic compound as the final electron acceptor

The fermentation process could be defined in different ways. If we think in the biochemical field, it breakdown the chemical bonds in sugars and converts into the energy which is not possible to produce in the glycolysis process.

During our normal activities, the amount of oxygen for breathing is enough in our body but when we involve in high activities in our daily life our body cannot supply enough oxygen for body cells, as a result, we breathe faster.

So, that time how body cells keep cellular respiration function without enough oxygen?

Without oxygen, the glycolysis process can be continued. If oxygen is available, it is used in cellular respiration where oxygen picks up the electrons. But if there is no oxygen electron couldn’t be possible to pick up and this time glycolysis processes can continue by the production of ATP without oxygen.

Figure: Fermentation Process.

ATP molecules are made in the glycolysis process. Nevertheless, the ATP molecules are not made in the fermentation process, but it allows glycolysis to continue. Fermentation can remove the electron from NADH molecules and regenerate NAD+ molecules which is need for glycolysis that picks up the electron where no need oxygen to pick up the electrons for continuing the body function. Glycolysis process would be stopped if there is no electron pick up and without NAD+ it is not possible to pick up the electrons from the splitting of glucose.

How NAD+ can help for continuing the glycolysis process?

When oxygen is not present in the cell, in the glycolysis process, glucose is broken down into two molecules of pyruvate by the production of two molecules of ATP and reduces NAD+ molecule into NADH which is a store of energy.

Then, during the fermentation process, two NADH molecules provide energy to convert pyruvate into fermentation products. By way of the NADH is used, it is rehabilitated back into NAD+. Two molecules of NAD+ are recycled back to glycolysis. Then the glycolysis process could be continuing by the recycled NAD+.

Please make comment, if you feel interesting this article or as any questions.

What you'll learn:

The process of glycolysis includes the conversion of the reactant, glucose plus two molecules NAD+, two molecules of ADP plus 2Pi, into the products, i.e., two molecules of pyruvate plus two molecules of NADH plus two molecules H+ and two molecules of ATP. If the glycolysis occurred continuously, all the molecules NAD+ would have been used entirely, and this would end up the cycle of glycolysis. To continue the cycle of glycolysis, there must occur the conversion of NADH back to NAD+. The occurrence of this step is based on the available external electron acceptor. The first method that can be used to carry out the same is the conversion of pyruvate to lactate, and this process is termed lactic acid fermentation. In this reaction, the pyruvate, NADH, and H+ is the reactant that gets converted into lactate and NAD+. This process also happens within the bacteria that are used to make yogurt. There are individual organisms, for example, yeast, which involves the conversion of NADH to NAD+ through the process termed as ethanol fermentation. There occurs the conversion of pyruvate into acetaldehyde and carbon dioxide, which in turn is converted into ethanol. Both ethanol fermentation and lactic acid fermentation takes place in the absence of Oxygen. Therefore such anaerobic fermentation can be e used by single-celled organisms as their source of energy. Anoxic regeneration NAD+ can be studied as an effective means for the production of energy during a short period that is from 10 seconds to about 2 minutes. There occurs the replenishment of NAD+ through NADH by giving the electrons to the pyruvate, which in turn leads to the production of lactate. This ultimately produces two ATP molecules that are formed from one glucose molecule. Fermentation of pyruvate into lactate can also be termed as anaerobic glycolysis. In the mention of two fermentation, the oxidation of NADH took place, which leads to the transfer of two electrons to pyruvate.

How NAD+ Is Powerful

Open any biology textbook and you’ll learn about NAD+, which stands for nicotinamide adenine dinucleotide. It’s a critical coenzyme found in every cell in your body that’s involved in hundreds of metabolic processes like cellular energy and mitochondrial health. NAD+ is hard at work in the cells of humans and other mammals, yeast and bacteria, even plants.

Scientists have known about NAD+ since it was first discovered in 1906, and since then our understanding of its importance has continued to evolve. For example, the NAD+ precursor niacin played a role in mitigating pellagra, a fatal disease that plagued the American south in the 1900s. Scientists at the time identified that milk and yeast, which both contain NAD+ precursors, alleviated symptoms. Over time scientists have identified several NAD+ precursors — including nicotinic acid, nicotinamide, and nicotinamide riboside, among others — which make use of natural pathways that lead to NAD+. Think of NAD+ precursors as different routes you can take to get to a destination. All the pathways get you to the same place but by different modes of transportation.

Recently, NAD+ has become a prized molecule in scientific research because of its central role in biological functions. The scientific community has been researching how NAD+ relates to notable benefits in animals that continue to inspire researchers to translate these findings to humans. So how exactly does NAD+ play such an important role? In short, it’s a coenzyme or “helper” molecule, binding to other enzymes to help cause reactions on the molecular level.

Increased demand for NAD + relative to ATP drives aerobic glycolysis

Aerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is associated with proliferation across many organisms and conditions. To better understand that association, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the NAD + /NADH ratio. This change in NAD + /NADH is caused by increased mitochondrial membrane potential that impairs mitochondrial electron transport and NAD + regeneration. Uncoupling respiration from ATP synthesis or increasing ATP hydrolysis restores NAD + /NADH homeostasis and proliferation even when glucose oxidation is increased. These data suggest that when demand for NAD + to support oxidation reactions exceeds the rate of ATP turnover in cells, NAD + regeneration by mitochondrial respiration becomes constrained, promoting fermentation, despite available oxygen. This argues that cells engage in aerobic glycolysis when the demand for NAD + is in excess of the demand for ATP.

Keywords: Aerobic Glycolysis Cell Metabolism Fermentation NAD+ PDK Warburg Effect.

Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

The Evolution and the Development Phases of Wine

Monica Butnariu , Alina Butu , in Alcoholic Beverages , 2019

10.11 Alcoholic Fermentation

Alcoholic fermentation of the must is a spontaneous or induced biochemical oxidoreduction process by which, under the action of yeast enzymes, carbohydrates convert to ethyl alcohol and CO 2 as the main products accompanied by several by-products.

This process is exothermic, and for achieving one degree of alcohol between 15.7 and 18 g of sugar/L must be used. The decomposition of carbohydrates into alcohol and CO2 takes place inside yeast cells. The sugar solution penetrates the cells membrane, and the resulting products (alcohol, CO2, etc.) are diffused in the environment. The process is determined by yeast activity as they contain the enzymes needed to carry out the fermentation.

Under the action of the yeast enzyme complex, the sugar in the must turns into a phosphoglyceral aldehyde, after absorption into the cells and formation of the phosphoric esters. By oxidation-reduction reactions and quantified release of potential energy, 2 mol of ATP, one mole of triose, and the main products of fermentation are formed. These include CO2, obtained by decarboxylation of pyruvic acid and ethyl alcohol, obtained by reduction of acetic aldehyde in the presence of dehydrogenase.

10.11.1 Enzymatic Nature of the Fermentation Process

Alcoholic fermentation is a complex process in which enzymes act as a catalyst for carbohydrate decomposition reactions and the formation of specific compounds. The enzyme classes of yeast include: oxidoreductases, hydrolase, transferases, lysates, isomerases, ligases, and synthases. In the fermentation process the following enzymes are involved: hexokinase, aldolase, dehydrogenase, phosphohexoisomerase, phosphohexokinase, triose isomerase, pyruvate kinase, pyruvate decarboxylase, aldohydrogenase, etc. Enzymes interfere in the fermentation process in successive stages, acting specifically, through their active components, coenzymes.

10.11.2 Coenzymes Participating in the Fermentation Process

Nicotinamide-adenine-dinucleotide (NAD +) is the coenzyme of many enzymes in the dehydrogenase class, the role is to reversibly fix the hydrogen ions delivered to the substrate, and the oxidoreduction mechanism takes place at the level of the pyridine nucleus. It is oxidized in the presence of a positive charge from the nitrogen atom, or reduced if there is no such nitrogen load. Cocarboxylase or thiamine pyrophosphate (TPP) is the enzyme coenzyme in the decarboxylase class to decarboxylate ketones to aldehydes in the presence of Mg 2 + ions.

Adenosine triphosphate (ATP) is involved in the transport of phosphate ions, that is, in the phosphorylation of carbohydrates, with a role in the energy balance of fermentation.

Adenosine diphosphate (ADP) is involved in the transport of phosphate ions, that is, phosphorylation of carbohydrates. Coenzyme A (CoA-SH) is an amide compound of pantothenic acid. Its activity is imprinted by the acetyl-linked SH group in the form of thioester (acetyl coenzyme A). The reaction is rich in energy and is coupled with the formation of the energy-rich ATP molecule.

10.11.3 Biochemical Mechanism of Alcoholic Fermentation

The process of fermentation is a process of chemical degradation under the action of enzymes, of natural products with complex structures in products with simple structure. This process generates energy (caloric energy). These are the steps of a complete fermentation cycle:

the biomass accumulation stage, when the fermentation is reduced

the main fermentation, about 80% of the initial sugar is fermented and

secondary fermentation when alcohols are formed.

Alcoholic fermentation takes place after the yeast glycolysis biochemical mechanism by which hexoses are converted to pyruvic acid and subsequently into ethyl alcohol and CO 2.

Higher alcohols (propyl alcohol, isopropyl alcohol, isobutyl alcohol, amyl alcohol, isoamyl alcohol) are formed in the wine and form the bouquet during wine enrichment, improving olfactory-taste qualities through the appearance of the esters. Glycerol (glycerin) is a by-product of alcoholic fermentation that is formed at the beginning of the process. The proportion of glycerol in wine depends on:

the initial concentration of must in carbohydrates

the amount of SO2 used to protect the must

temperature maintained during fermentation

the duration of the alcoholic fermentation process and

yeasts that have carried out alcoholic fermentation, etc. ( Springer et al., 2016 ).

After these conditions, glycerol is found in the proportion of 6–10 g for each 100 g of ethyl alcohol. Wines where the glycerol/alcohol ratio is below 6.5% were previously alcoholized and when this ratio exceeds 10%, wines may be suspected of having been added glycerol. Natural wines have glycerol content between 5 and 15 g/L. Acetic aldehyde accumulates in the first 2–3 days of fermentation, and the concentration varies between 40 and 50 mg/L. Aromatic aldehydes (benzoic aldehyde, vanillin, cinnamic aldehyde, acetone, diacetyl) are formed. They are needed in the synthesis of the flavor and bouquet characteristic of wines. In the wine, volatile acids, dependent on the predominant yeast species, are formed, between 10 and 280 mg/L. In addition to malic acid, tartaric acid, and citric acid, 10–16 mg/L pyrogenic acid is accumulated by fermentation, α-ketoglutaric acid, 90–119 mg/L, acetic acid, lactic acid, etc. Most flavorings that are formed during fermentation are produced from yeast nitrate metabolism and are a consequence of imperfect coordination of enzyme activity involved in these biochemical processes ( Goold et al., 2017 ).

10.11.4 Sulfur Metabolization by Yeast

The growth and multiplication of yeasts is conditioned by the presence in the must of assimilable sulfur sources such as sulfates and small amounts of biotin and thiamine. Part of the yeast consumes sulfur from methionine, because cystine and cysteine are degraded hard and are difficult to assimilate. From the chemical composition of the yeast, 0.2%–0.8% of the dry substance is sulfur it enters the structure of proteins and enzymatic cofactors (biotin, thiamine, lipoic acid, etc.). Yeast cells reduce sulfates to sulfites and H2S, used for sulfur biosynthesis. During storage of yeast wine, following autolysis, H2S is formed by the action of cysteine sulfhydrylase acting on nonvalent compounds with –SH groups. It negatively influences the quality of wine, forming ethyl mercaptans, which imparts unwanted taste and odor. By metabolizing the sulfur compounds, the yeast can produce 10–80 mg SO2/L at the end of the fermentation period. To stabilize young wines, special yeast cultures, which produce up to 80 mg SO2/L during fermentation, are used to prevent oxidative decomposition.

10.11.5 Must Fermentation Technology

The required operations are the preparation of selected yeast starter cultures filling fermentation containers with must inoculation with selected yeasts addition of fermentation activators managing the fermentation process and interruption of fermentation for sweet wines.

10.11.6 Preparation of Yeast Starter Cultures

To obtain different types of high-quality wines, the fermentation is carried out by obtaining selected cultures of strains of the genus Saccharomyces ellipsoideus and Saccharomyces oviformis. These yeasts create favorable conditions for the fermentation of must and the activity of other yeasts.

The optimum temperature of fermentation is between 22°C and 27°C. For a favorable development, the yeast of the must depends on: the temperature that increases as the quantity of ethyl alcohol and CO2 increases, reaching up to 35°C osmotic pressure of the must oxidation-reduction potential of the must the level of nitrogen in the must the concentration of CO2 and oxygen volatile acids (formic, acetic, propionic, lactic) tanning substances and mineral salts and vitamins in the must. The preparation of selected yeast starter cultures in the form of active leaven is carried out in special plants.

The operation is done 1 week prior to the beginning of the grape vinification campaign, first at laboratory level, and then going through production stage. The laboratory stage is more important because the quality of the wine will depend on the yeast strain used. The yeast is selected, preserved, and multiplied in optimum conditions. In the production stage the selected yeast culture will be transformed to leaven ( Ciani et al., 2016 ).

Chapter 9 – Cellular Respiration

· To perform their many tasks, living cells require energy from outside sources.

· Energy enters most ecosystems as sunlight and leaves as heat.

· Photosynthesis generates oxygen and organic molecules that the mitochondria of eukaryotes use as fuel for cellular respiration.

· Cells harvest the chemical energy stored in organic molecules and use it to regenerate ATP, the molecule that drives most cellular work.

· Respiration has three key pathways: glycolysis, the citric acid cycle, and oxidative phosphorylation.

A. The Principles of Energy Harvest

1. Cellular respiration and fermentation are catabolic, energy-yielding pathways.

· The arrangement of atoms of organic molecules represents potential energy.

· Enzymes catalyze the systematic degradation of organic molecules that are rich in energy to simpler waste products with less energy.

· Some of the released energy is used to do work the rest is dissipated as heat.

· Catabolic metabolic pathways release the energy stored in complex organic molecules.

· One type of catabolic process, fermentation, leads to the partial degradation of sugars in the absence of oxygen.

· A more efficient and widespread catabolic process, cellular respiration, consumes oxygen as a reactant to complete the breakdown of a variety of organic molecules.

° In eukaryotic cells, mitochondria are the site of most of the processes of cellular respiration.

· Cellular respiration is similar in broad principle to the combustion of gasoline in an automobile engine after oxygen is mixed with hydrocarbon fuel.

° Food is the fuel for respiration. The exhaust is carbon dioxide and water.

° organic compounds + O2 à CO2 + H2O + energy (ATP + heat).

· Carbohydrates, fats, and proteins can all be used as the fuel, but it is most useful to consider glucose.

° C6H12O6 + 6O2 à 6CO2 + 6H2O + Energy (ATP + heat)

· The catabolism of glucose is exergonic with a D G of −686 kcal per mole of glucose.

° Some of this energy is used to produce ATP, which can perform cellular work.

2. Redox reactions release energy when electrons move closer to electronegative atoms.

· Catabolic pathways transfer the electrons stored in food molecules, releasing energy that is used to synthesize ATP.

· Reactions that result in the transfer of one or more electrons from one reactant to another are oxidation-reduction reactions, or redox reactions.

° The loss of electrons is called oxidation.

° The addition of electrons is called reduction.

· The formation of table salt from sodium and chloride is a redox reaction.

° Here sodium is oxidized and chlorine is reduced (its charge drops from 0 to −1).

· More generally: Xe− + Y à X + Ye−

° X, the electron donor, is the reducing agent and reduces Y.

° Y, the electron recipient, is the oxidizing agent and oxidizes X.

· Redox reactions require both a donor and acceptor.

· Redox reactions also occur when the transfer of electrons is not complete but involves a change in the degree of electron sharing in covalent bonds.

° In the combustion of methane to form water and carbon dioxide, the nonpolar covalent bonds of methane (C—H) and oxygen (O=O) are converted to polar covalent bonds (C=O and O—H).

° When methane reacts with oxygen to form carbon dioxide, electrons end up farther away from the carbon atom and closer to their new covalent partners, the oxygen atoms, which are very electronegative.

° In effect, the carbon atom has partially “lost” its shared electrons. Thus, methane has been oxidized.

· The two atoms of the oxygen molecule share their electrons equally. When oxygen reacts with the hydrogen from methane to form water, the electrons of the covalent bonds are drawn closer to the oxygen.

° In effect, each oxygen atom has partially “gained” electrons, and so the oxygen molecule has been reduced.

° Oxygen is very electronegative, and is one of the most potent of all oxidizing agents.

· Energy must be added to pull an electron away from an atom.

· The more electronegative the atom, the more energy is required to take an electron away from it.

· An electron loses potential energy when it shifts from a less electronegative atom toward a more electronegative one.

· A redox reaction that relocates electrons closer to oxygen, such as the burning of methane, releases chemical energy that can do work.

3. The “fall” of electrons during respiration is stepwise, via NAD+ and an electron transport chain.

· Cellular respiration does not oxidize glucose in a single step that transfers all the hydrogen in the fuel to oxygen at one time.

· Rather, glucose and other fuels are broken down in a series of steps, each catalyzed by a specific enzyme.

° At key steps, electrons are stripped from the glucose.

° In many oxidation reactions, the electron is transferred with a proton, as a hydrogen atom.

· The hydrogen atoms are not transferred directly to oxygen but are passed first to a coenzyme called NAD+ (nicotinamide adenine dinucleotide).

· How does NAD+ trap electrons from glucose?

° Dehydrogenase enzymes strip two hydrogen atoms from the fuel (e.g., glucose), oxidizing it.

° The enzyme passes two electrons and one proton to NAD+.

° The other proton is released as H+ to the surrounding solution.

· By receiving two electrons and only one proton, NAD+ has its charge neutralized when it is reduced to NADH.

° NAD+ functions as the oxidizing agent in many of the redox steps during the catabolism of glucose.

· The electrons carried by NADH have lost very little of their potential energy in this process.

· Each NADH molecule formed during respiration represents stored energy. This energy is tapped to synthesize ATP as electrons “fall” from NADH to oxygen.

· How are electrons extracted from food and stored by NADH finally transferred to oxygen?

° Unlike the explosive release of heat energy that occurs when H2 and O2 are combined (with a spark for activation energy), cellular respiration uses an electron transport chain to break the fall of electrons to O2 into several steps.

· The electron transport chain consists of several molecules (primarily proteins) built into the inner membrane of a mitochondrion.

· Electrons released from food are shuttled by NADH to the “top” higher-energy end of the chain.

· At the “bottom” lower-energy end, oxygen captures the electrons along with H+ to form water.

· Electron transfer from NADH to oxygen is an exergonic reaction with a free energy change of −53 kcal/mol.

· Electrons are passed to increasingly electronegative molecules in the chain until they reduce oxygen, the most electronegative receptor.

· In summary, during cellular respiration, most electrons travel the following “downhill” route: food à NADH à electron transport chain à oxygen.

B. The Process of Cellular Respiration

1. These are the stages of cellular respiration: a preview.

· Respiration occurs in three metabolic stages: glycolysis, the citric acid cycle, and the electron transport chain and oxidative phosphorylation.

· Glycolysis occurs in the cytoplasm.

° It begins catabolism by breaking glucose into two molecules of pyruvate.

· The citric acid cycle occurs in the mitochondrial matrix.

° It completes the breakdown of glucose by oxidizing a derivative of pyruvate to carbon dioxide.

· Several steps in glycolysis and the citric acid cycle are redox reactions in which dehydrogenase enzymes transfer electrons from substrates to NAD+, forming NADH.

· NADH passes these electrons to the electron transport chain.

· In the electron transport chain, the electrons move from molecule to molecule until they combine with molecular oxygen and hydrogen ions to form water.

· As they are passed along the chain, the energy carried by these electrons is transformed in the mitochondrion into a form that can be used to synthesize ATP via oxidative phosphorylation.

· The inner membrane of the mitochondrion is the site of electron transport and chemiosmosis, processes that together constitute oxidative phosphorylation.

° Oxidative phosphorylation produces almost 90% of the ATP generated by respiration.

· Some ATP is also formed directly during glycolysis and the citric acid cycle by substrate-level phosphorylation.

° Here an enzyme transfers a phosphate group from an organic substrate to ADP, forming ATP.

· For each molecule of glucose degraded to carbon dioxide and water by respiration, the cell makes up to 38 ATP, each with 7.3 kcal/mol of free energy.

· Respiration uses the small steps in the respiratory pathway to break the large denomination of energy contained in glucose into the small change of ATP.

° The quantity of energy in ATP is more appropriate for the level of work required in the cell.

2. Glycolysis harvests chemical energy by oxidizing glucose to pyruvate.

· During glycolysis, glucose, a six carbon-sugar, is split into two three-carbon sugars.

· These smaller sugars are oxidized and rearranged to form two molecules of pyruvate, the ionized form of pyruvic acid.

· Each of the ten steps in glycolysis is catalyzed by a specific enzyme.

· These steps can be divided into two phases: an energy investment phase and an energy payoff phase.

· In the energy investment phase, the cell invests ATP to provide activation energy by phosphorylating glucose.

° This requires 2 ATP per glucose.

· In the energy payoff phase, ATP is produced by substrate-level phosphorylation and NAD+ is reduced to NADH by electrons released by the oxidation of glucose.

· The net yield from glycolysis is 2 ATP and 2 NADH per glucose.

° No CO2 is produced during glycolysis.

· Glycolysis can occur whether O2 is present or not.

3. The citric acid cycle completes the energy-yielding oxidation of organic molecules.

· More than three-quarters of the original energy in glucose is still present in the two molecules of pyruvate.

· If oxygen is present, pyruvate enters the mitochondrion where enzymes of the citric acid cycle complete the oxidation of the organic fuel to carbon dioxide.

· After pyruvate enters the mitochondrion via active transport, it is converted to a compound called acetyl coenzyme A or acetyl CoA.

· This step is accomplished by a multienzyme complex that catalyzes three reactions:

1. A carboxyl group is removed as CO2.

2. The remaining two-carbon fragment is oxidized to form acetate. An enzyme transfers the pair of electrons to NAD+ to form NADH.

3. Acetate combines with coenzyme A to form the very reactive molecule acetyl CoA.

· Acetyl CoA is now ready to feed its acetyl group into the citric acid cycle for further oxidation.

· The citric acid cycle is also called the Krebs cycle in honor of Hans Krebs, who was largely responsible for elucidating its pathways in the 1930s.

· The citric acid cycle oxidizes organic fuel derived from pyruvate.

° The citric acid cycle has eight steps, each catalyzed by a specific enzyme.

° The acetyl group of acetyl CoA joins the cycle by combining with the compound oxaloacetate, forming citrate.

° The next seven steps decompose the citrate back to oxaloacetate. It is the regeneration of oxaloacetate that makes this process a cycle.

° Three CO2 molecules are released, including the one released during the conversion of pyruvate to acetyl CoA.

· The cycle generates one ATP per turn by substrate-level phosphorylation.

° A GTP molecule is formed by substrate-level phosphorylation.

° The GTP is then used to synthesize an ATP, the only ATP generated directly by the citric acid cycle.

· Most of the chemical energy is transferred to NAD+ and FAD during the redox reactions.

· The reduced coenzymes NADH and FADH2 then transfer high-energy electrons to the electron transport chain.

· Each cycle produces one ATP by substrate-level phosphorylation, three NADH, and one FADH2 per acetyl CoA.

4. The inner mitochondrial membrane couples electron transport to ATP synthesis.

· Only 4 of 38 ATP ultimately produced by respiration of glucose are produced by substrate-level phosphorylation.

° Two are produced during glycolysis, and 2 are produced during the citric acid cycle.

· NADH and FADH2 account for the vast majority of the energy extracted from the food.

° These reduced coenzymes link glycolysis and the citric acid cycle to oxidative phosphorylation, which uses energy released by the electron transport chain to power ATP synthesis.

· The electron transport chain is a collection of molecules embedded in the cristae, the folded inner membrane of the mitochondrion.

° The folding of the cristae increases its surface area, providing space for thousands of copies of the chain in each mitochondrion.

° Most components of the chain are proteins bound to prosthetic groups, nonprotein components essential for catalysis.

· Electrons drop in free energy as they pass down the electron transport chain.

· During electron transport along the chain, electron carriers alternate between reduced and oxidized states as they accept and donate electrons.

° Each component of the chain becomes reduced when it accepts electrons from its “uphill” neighbor, which is less electronegative.

° It then returns to its oxidized form as it passes electrons to its more electronegative “downhill” neighbor.

· Electrons carried by NADH are transferred to the first molecule in the electron transport chain, a flavoprotein.

· The electrons continue along the chain that includes several cytochrome proteins and one lipid carrier.

° The prosthetic group of each cytochrome is a heme group with an iron atom that accepts and donates electrons.

· The last cytochrome of the chain, cyt a3, passes its electrons to oxygen, which is very electronegative.

° Each oxygen atom also picks up a pair of hydrogen ions from the aqueous solution to form water.

° For every two electron carriers (four electrons), one O2 molecule is reduced to two molecules of water.

· The electrons carried by FADH2 have lower free energy and are added at a lower energy level than those carried by NADH.

° The electron transport chain provides about one-third less energy for ATP synthesis when the electron donor is FADH2 rather than NADH.

· The electron transport chain generates no ATP directly.

· Its function is to break the large free energy drop from food to oxygen into a series of smaller steps that release energy in manageable amounts.

· How does the mitochondrion couple electron transport and energy release to ATP synthesis?

° The answer is a mechanism called chemiosmosis.

· A protein complex, ATP synthase, in the cristae actually makes ATP from ADP and Pi.

· ATP uses the energy of an existing proton gradient to power ATP synthesis.

° The proton gradient develops between the intermembrane space and the matrix.

· The proton gradient is produced by the movement of electrons along the electron transport chain.

· The chain is an energy converter that uses the exergonic flow of electrons to pump H+ from the matrix into the intermembrane space.

· The protons pass back to the matrix through a channel in ATP synthase, using the exergonic flow of H+ to drive the phosphorylation of ADP.

· Thus, the energy stored in a H+ gradient across a membrane couples the redox reactions of the electron transport chain to ATP synthesis.

· From studying the structure of ATP synthase, scientists have learned how the flow of H+ through this large enzyme powers ATP generation.

· ATP synthase is a multisubunit complex with four main parts, each made up of multiple polypeptides:

1. A rotor in the inner mitochondrial membrane.

2. A knob that protrudes into the mitochondrial matrix.

3. An internal rod extending from the rotor into the knob.

4. A stator, anchored next to the rotor, which holds the knob stationary.

· Protons flow down a narrow space between the stator and rotor, causing the rotor and its attached rod to rotate.

° The spinning rod causes conformational changes in the stationary knob, activating three catalytic sites in the knob where ADP and inorganic phosphate combine to make ATP.

· How does the inner mitochondrial membrane generate and maintain the H+ gradient that drives ATP synthesis in the ATP synthase protein complex?

° Creating the H+ gradient is the function of the electron transport chain.

° The ETC is an energy converter that uses the exergonic flow of electrons to pump H+ across the membrane from the mitochondrial matrix to the intermembrane space.

° The H+ has a tendency to diffuse down its gradient.

· The ATP synthase molecules are the only place that H+ can diffuse back to the matrix.

° The exergonic flow of H+ is used by the enzyme to generate ATP.

° This coupling of the redox reactions of the electron transport chain to ATP synthesis is called chemiosmosis.

· How does the electron transport chain pump protons?

° Certain members of the electron transport chain accept and release H+ along with electrons.

° At certain steps along the chain, electron transfers cause H+ to be taken up and released into the surrounding solution.

· The electron carriers are spatially arranged in the membrane in such a way that protons are accepted from the mitochondrial matrix and deposited in the intermembrane space.

° The H+ gradient that results is the proton-motive force.

° The gradient has the capacity to do work.

· Chemiosmosis is an energy-coupling mechanism that uses energy stored in the form of an H+ gradient across a membrane to drive cellular work.

· In mitochondria, the energy for proton gradient formation comes from exergonic redox reactions, and ATP synthesis is the work performed.

· Chemiosmosis in chloroplasts also generates ATP, but light drives the electron flow down an electron transport chain and H+ gradient formation.

· Prokaryotes generate H+ gradients across their plasma membrane.

° They can use this proton-motive force not only to generate ATP, but also to pump nutrients and waste products across the membrane and to rotate their flagella.

5. Here is an accounting of ATP production by cellular respiration.

· During cellular respiration, most energy flows from glucose à NADH à electron transport chain à proton-motive force à ATP.

· Let’s consider the products generated when cellular respiration oxidizes a molecule of glucose to six CO2 molecules.

· Four ATP molecules are produced by substrate-level phosphorylation during glycolysis and the citric acid cycle.

· Many more ATP molecules are generated by oxidative phosphorylation.

· Each NADH from the citric acid cycle and the conversion of pyruvate contributes enough energy to the proton-motive force to generate a maximum of 3 ATP.

° The NADH from glycolysis may also yield 3 ATP.

· Each FADH2 from the citric acid cycle can be used to generate about 2 ATP.

· Why is our accounting so inexact?

· There are three reasons that we cannot state an exact number of ATP molecules generated by one molecule of glucose.

1. Phosphorylation and the redox reactions are not directly coupled to each other, so the ratio of number of NADH to number of ATP is not a whole number.

° One NADH results in 10 H+ being transported across the inner mitochondrial membrane.

° Between 3 and 4 H+ must reenter the mitochondrial matrix via ATP synthase to generate 1 ATP.

° Therefore, 1 NADH generates enough proton-motive force for synthesis of 2.5 to 3.3 ATP.

° We round off and say that 1 NADH generates 3 ATP.

2. The ATP yield varies slightly depending on the type of shuttle used to transport electrons from the cytosol into the mitochondrion.

° The mitochondrial inner membrane is impermeable to NADH, so the two electrons of the NADH produced in glycolysis must be conveyed into the mitochondrion by one of several electron shuttle systems.

° In some shuttle systems, the electrons are passed to NAD+, which generates 3 ATP. In others, the electrons are passed to FAD, which generates only 2 ATP.

3. The proton-motive force generated by the redox reactions of respiration may drive other kinds of work, such as mitochondrial uptake of pyruvate from the cytosol.

° If all the proton-motive force generated by the electron transport chain were used to drive ATP synthesis, one glucose molecule could generate a maximum of 34 ATP by oxidative phosphorylation plus 4 ATP (net) from substrate-level phosphorylation to give a total yield of 36–38 ATP (depending on the efficiency of the shuttle).

· How efficient is respiration in generating ATP?

° Complete oxidation of glucose releases 686 kcal/mol.

° Phosphorylation of ADP to form ATP requires at least 7.3 kcal/mol.

° Efficiency of respiration is 7.3 kcal/mol times 38 ATP/glucose divided by 686 kcal/mol glucose, which equals 0.4 or 40%.

° Approximately 60% of the energy from glucose is lost as heat.

§ Some of that heat is used to maintain our high body temperature (37°C).

· Cellular respiration is remarkably efficient in energy conversion.

C. Related Metabolic Processes

1. Fermentation enables some cells to produce ATP without the help of oxygen.

· Without electronegative oxygen to pull electrons down the transport chain, oxidative phosphorylation ceases.

· However, fermentation provides a mechanism by which some cells can oxidize organic fuel and generate ATP without the use of oxygen.

° In glycolysis, glucose is oxidized to two pyruvate molecules with NAD+ as the oxidizing agent.

° Glycolysis is exergonic and produces 2 ATP (net).

° If oxygen is present, additional ATP can be generated when NADH delivers its electrons to the electron transport chain.

· Glycolysis generates 2 ATP whether oxygen is present (aerobic) or not (anaerobic).

· Anaerobic catabolism of sugars can occur by fermentation.

· Fermentation can generate ATP from glucose by substrate-level phosphorylation as long as there is a supply of NAD+ to accept electrons.

° If the NAD+ pool is exhausted, glycolysis shuts down.

° Under aerobic conditions, NADH transfers its electrons to the electron transfer chain, recycling NAD+.

· Under anaerobic conditions, various fermentation pathways generate ATP by glycolysis and recycle NAD+ by transferring electrons from NADH to pyruvate or derivatives of pyruvate.

· In alcohol fermentation, pyruvate is converted to ethanol in two steps.

° First, pyruvate is converted to a two-carbon compound, acetaldehyde, by the removal of CO2.

° Second, acetaldehyde is reduced by NADH to ethanol.

° Alcohol fermentation by yeast is used in brewing and winemaking.

· During lactic acid fermentation, pyruvate is reduced directly by NADH to form lactate (the ionized form of lactic acid) without release of CO2.

° Lactic acid fermentation by some fungi and bacteria is used to make cheese and yogurt.

° Human muscle cells switch from aerobic respiration to lactic acid fermentation to generate ATP when O2 is scarce.

§ The waste product, lactate, may cause muscle fatigue, but ultimately it is converted back to pyruvate in the liver.

· Fermentation and cellular respiration are anaerobic and aerobic alternatives, respectively, for producing ATP from sugars.

° Both use glycolysis to oxidize sugars to pyruvate with a net production of 2 ATP by substrate-level phosphorylation.

° Both use NAD+ as an oxidizing agent to accept electrons from food during glycolysis.

· The two processes differ in their mechanism for oxidizing NADH to NAD+.

° In fermentation, the electrons of NADH are passed to an organic molecule to regenerate NAD+.

° In respiration, the electrons of NADH are ultimately passed to O2, generating ATP by oxidative phosphorylation.

· More ATP is generated from the oxidation of pyruvate in the citric acid cycle.

° Without oxygen, the energy still stored in pyruvate is unavailable to the cell.

° Under aerobic respiration, a molecule of glucose yields 38 ATP, but the same molecule of glucose yields only 2 ATP under anaerobic respiration.

· Yeast and many bacteria are facultative anaerobes that can survive using either fermentation or respiration.

° At a cellular level, human muscle cells can behave as facultative anaerobes.

· For facultative anaerobes, pyruvate is a fork in the metabolic road that leads to two alternative routes.

° Under aerobic conditions, pyruvate is converted to acetyl CoA and oxidation continues in the citric acid cycle.

° Under anaerobic conditions, pyruvate serves as an electron acceptor to recycle NAD+.

· The oldest bacterial fossils are more than 3.5 billion years old, appearing long before appreciable quantities of O2 accumulated in the atmosphere.

° Therefore, the first prokaryotes may have generated ATP exclusively from glycolysis.

· The fact that glycolysis is a ubiquitous metabolic pathway and occurs in the cytosol without membrane-enclosed organelles suggests that glycolysis evolved early in the history of life.

2. Glycolysis and the citric acid cycle connect to many other metabolic pathways.

· Glycolysis can accept a wide range of carbohydrates for catabolism.

° Polysaccharides like starch or glycogen can be hydrolyzed to glucose monomers that enter glycolysis.

° Other hexose sugars, such as galactose and fructose, can also be modified to undergo glycolysis.

· The other two major fuels, proteins and fats, can also enter the respiratory pathways used by carbohydrates.

· Proteins must first be digested to individual amino acids.

° Amino acids that will be catabolized must have their amino groups removed via deamination.

° The nitrogenous waste is excreted as ammonia, urea, or another waste product.

· The carbon skeletons are modified by enzymes and enter as intermediaries into glycolysis or the citric acid cycle, depending on their structure.

· Catabolism can also harvest energy stored in fats.

· Fats must be digested to glycerol and fatty acids.

° Glycerol can be converted to glyceraldehyde phosphate, an intermediate of glycolysis.

° The rich energy of fatty acids is accessed as fatty acids are split into two-carbon fragments via beta oxidation.

° These molecules enter the citric acid cycle as acetyl CoA.

· A gram of fat oxides by respiration generates twice as much ATP as a gram of carbohydrate.

· The metabolic pathways of respiration also play a role in anabolic pathways of the cell.

· Intermediaries in glycolysis and the citric acid cycle can be diverted to anabolic pathways.

° For example, a human cell can synthesize about half the 20 different amino acids by modifying compounds from the citric acid cycle.

° Glucose can be synthesized from pyruvate fatty acids can be synthesized from acetyl CoA.

· Glycolysis and the citric acid cycle function as metabolic interchanges that enable cells to convert one kind of molecule to another as needed.

° For example, excess carbohydrates and proteins can be converted to fats through intermediaries of glycolysis and the citric acid cycle.

· Metabolism is remarkably versatile and adaptable.

3. Feedback mechanisms control cellular respiration.

· Basic principles of supply and demand regulate the metabolic economy.

° If a cell has an excess of a certain amino acid, it typically uses feedback inhibition to prevent the diversion of intermediary molecules from the citric acid cycle to the synthesis pathway of that amino acid.

· The rate of catabolism is also regulated, typically by the level of ATP in the cell.

° If ATP levels drop, catabolism speeds up to produce more ATP.

· Control of catabolism is based mainly on regulating the activity of enzymes at strategic points in the catabolic pathway.

· One strategic point occurs in the third step of glycolysis, catalyzed by phosphofructokinase.

· Allosteric regulation of phosphofructokinase sets the pace of respiration.

° This enzyme catalyzes the earliest step that irreversibly commits the substrate to glycolysis.

° Phosphofructokinase is an allosteric enzyme with receptor sites for specific inhibitors and activators.

° It is inhibited by ATP and stimulated by AMP (derived from ADP).

§ When ATP levels are high, inhibition of this enzyme slows glycolysis.

§ As ATP levels drop and ADP and AMP levels rise, the enzyme becomes active again and glycolysis speeds up.

· Citrate, the first product of the citric acid cycle, is also an inhibitor of phosphofructokinase.

° This synchronizes the rate of glycolysis and the citric acid cycle.

· If intermediaries from the citric acid cycle are diverted to other uses (e.g., amino acid synthesis), glycolysis speeds up to replace these molecules.

· Metabolic balance is augmented by the control of other enzymes at other key locations in glycolysis and the citric acid cycle.

Watch the video: ATP u0026 Respiration: Crash Course Biology #7 (November 2021).