Regulation of the Krebs cycle

The Krebs cycle plays a central role in our metabolism. In all the classes I give about metabolism, the Krebs cycle is present...
As I mentioned in previous posts, this process is composed by 8 steps, 3 of which are catalyzed by regulatory enzymes. These enzymes are citrate synthase (1st reaction), isocitrate dehydrogenase (3rd Reaction) and alpha-ketoglutarate dehydrogenase (4th reaction).
In this post I will talk a little about the main activators and inhibitors of each. As you will see, there are many modulators that are common to more than one enzyme, which makes life easier for those who have to study this metabolic pathway. :)

Citrate synthase:

Inhibitors
Succinyl-CoA - it is an intermediate of Krebs cycle. More specifically, it is the 4th intermediate of Krebs cycle, that means, it is formed in a reaction after the reaction that we are considering. So if we have an accumulation of intermediates formed in further reactions, it makes sense that these may inhibit the initial reactions of the pathway in question, in this case the first.
Citrate - it is the product of the reaction, so it makes sense that it might inhibit its own synthesis.
ATP - the Krebs cycle is a catabolic pathway, ie, its main goal is to produce energy (ATP). If the cell already has energy, the process is inhibited.
NADH - The reasoning is equivalent to that made for the ATP. That is, the NADH has a high energy potential, since in cellular respiration it can lead to the production of ATP, therefore it is logical that NADH functions as an inhibitor of Krebs cycle.
Long Chain fatty acid-CoA - it is not completely understood the inhibitory role of the long chain fatty acids in the Krebs cycle, but it is believed that this property is related to the fact that they behave as detergents because they are amphipathic compounds consisting of one polar part (carboxylic group) and one part apolar part (hydrocarbon chain). Oleic acid (18 carbons and one double bond at carbon 9) appears to be the major fatty acid inhibitor of citate synthase.

Activators
ADP - ADP signals an energy deficit in the cell because it is produced when ATP is spent for energy. So it makes sense that it activates the Krebs cycle, because the main objective of this pathway is the production of energy.

Isocitrate dehydrogenase:

Inhibitors
Succinyl-CoA - the reasoning that was made for the citrate synthase applies in this situation.
ATP - the reasoning that was made for the citrate synthase applies in this situation.
NADH - the reasoning that was made for the citrate synthase applies in this situation.

Activators
ADP - the reasoning that was made for the citrate synthase applies in this situation.
Ca2 + (muscle) - as I mentioned in a previous post, about the regulation of pyruvate dehydrogenase complex, Ca2+ is an intracellular messenger whose concentration increases during muscle contraction. Therefore, in this context contracting cells will require energy, so catabolic processes and, in particular, the Krebs cycle, is activated.

Alpha-ketoglutarate dehydrogenase:

 Inhibitors
Succinyl-CoA - it is the product of the reaction so, it makes sense that this molecule may inhibit its own synthesis.
ATP - the reasoning that was made for the citrate synthase applies in this situation.
NADH - the reasoning that was made for the citrate synthase applies in this situation.

Activators
Ca2 + (muscle) - the reasoning that was made for isocitrate dehydrogenase applies in this situation.

Regulation of pyruvate oxidation (part 2)

The pyruvate dehydrogenase complex activity is regulated mainly by two distinct mechanisms – allosteric mechanisms and reversible covalent modification.

In fact, there are some allosteric modulators of the complex, which in this particular case belong to the class of negative modulators, ie, inhibitors of its catalytic activity:

- Acetyl-CoA – it is the product of the reaction, thus, it makes sense that the molecule of acetyl-CoA has an inhibitory effect on its own synthesis

- NADH – one of the products of the reaction is NADH, so the reasoning is equivalent to that carried over to the acetyl-CoA molecule. Furthermore, as already mentioned in other posts of this blog, NADH may be involved in ATP synthesis (in cellular respiration), so, its presence indicates a potential for a high energy state into the cell. Accordingly, and as the oxidation of pyruvate to acetyl-CoA is part of catabolism, whose main objective is to obtain energy, it makes sense that NADH inhibits catabolism and, in particular, this reaction.

Regarding the reversible covalent modification, this enzyme complex is inhibited by phosphorylation and activated by dephosphorylation. This process is mediated by two different enzymes… The one that phosphorylates is called pyruvate dehydrogenase kinase, whereas the one that dephosphorylates it is the pyruvate dehydrogenase phosphatase.

Factors that activate the kinase, leading to the phosphorylation of pyruvate dehydrogenase complex (ie, inhibitors of its catalytic activity):

- Acetyl-CoA and NADH – in addition to its direct effects on the pyruvate dehydrogenase complex, as allosteric inhibitors, these two molecules also trigger the phosphorylation of the complex, promoting its inhibition, which means that they can act, therefore, through two distinct mechanisms

Factors that inhibit the kinase, favoring the balance towards the dephosphorylated form of pyruvate dehydrogenase complex (ie, activating its catalytic activity):

- NAD⁺ – for this molecule it can be done the reverse rationale made for NADH. That is, the presence of NAD⁺ indicates an energy deficit on the cell, so it is needed to activate the catabolism to counteract this deficit.

- ADP – the reasoning is equivalent to the one mentioned above, as to say that the cell is accumulating ADP means that it is spending ATP. Thus, it will need to produce again ATP

- Pyruvate – pyruvate is the substrate of the reaction, and its presence will activate the pyruvate dehydrogenase complex by inhibiting the phosphorylation process (and thus its inhibition) of the pyruvate dehydrogenase complex

- Coenzyme A (CoA) – this cofactor plays a co-substrate role, so that its presence will affect catalytic activity of the complex in a similar manner to that described for pyruvate

Factors that activate the phosphatase, leading to dephosphorylation of pyruvate dehydrogenase complex (ie, activating its catalytic activity):

- Ca²⁺ - calcium ion is an important modulator of the metabolism. In this particular case, this ion acts (in the muscle) at the level of pyruvate dehydrogenase phosphatase, activating it (by promoting its dephosphorylation). Put simply, the calcium ion is an indicator of muscle contraction, so it makes perfect sense that in the context of working muscles, the catabolism becomes active, so that there is ATP available for the process

Regulation of pyruvate oxidation (part 1)

The pyruvate dehydrogenase complex is mainly regulated by two distinct mechanisms: allosteric regulation and reversible covalent modification. Both can act (and act indeed!) at the same time, and there are molecules (activators and inhibitors) involved in both processes simultaneously.

Activators of the pyruvate dehydrogenase complex

- AMP and ADP – AMP and ADP are two molecules that are obtained when ATP is used as a source of chemical energy (ATP can be cleaved either to ADP or AMP). Therefore, both molecules indicate a low energy state, so it makes sense that they function as activators of processes that allow for energy, the catabolic processes. Since the oxidation of pyruvate is part of the catabolism, this process is activated by AMP and ADP.
- CoA – this is one of the cofactors of the enzyme that appears included in the products (pyruvate is both decarboxylated, oxidized and combined with CoA). Thus, as it is one of the molecules that will react with the substrate, its presence activates the enzyme.
- NAD+ – like the molecule of CoA, NAD+ is also used in the reaction, where it appears in the products (in the form of NADH). Furthermore, since the NADH can be used to promote ATP synthesis (in the cellular respiration), where it is oxidized to NAD+, the latter is an indicator of a low energy state in the cell. For all of this, it makes sense that this molecule is an activator of pyruvate oxidation.
- Ca2+ (muscle) – the calcium ion is an important mediator of various cellular responses. One of them is muscle contraction. Therefore, as this ion is an indicator of muscle contraction, which is an ATP-consuming process, it is advantageous for muscle cell that it can be simultaneously used as an activator of catabolism and, in particular of the oxidation of pyruvate. Thus, with the same messenger, muscle performs contraction and activates catabolism.
- Pyruvate – pyruvate is the substrate of the pyruvate dehydrogenase complex, so it makes sense that it can act as an activator.
- Dephosphorylation – complex pyruvate dehydrogenase is active in its dephosphorylated form.

Inhibitors of pyruvate dehydrogenase complex

- ATP – the main goal of catabolism is to produce energy, mainly in the form of ATP. If the cell has already ATP, or NADH (which, as mentioned above, can lead to the production of ATP), catabolism is inhibited.
- Acetyl-CoA – this is the product of the reaction, so it is natural that it has an inhibitory role in the process.
- Long chain fatty acids - some fatty acids, particularly those with long chains, can act as inhibitors of this reaction.
- Phosphorylation - pyruvate dehydrogenase complex is inactivated by reversible phosphorylation.
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Animation about feedback inhibition

Here it is the link to download a flash animation about feedback inhibition, also called negative feedback or retro-inhibition.

http://www.mediafire.com/?du7e5ag5poj2c4o

Metabolic map on the regulation of protein translation

http://www.biocarta.com/genes/Metabolism.asp

Regulation of glycolysis

Glycolysis provides different cell molecules important for cellular proper functioning. The main donor of chemical energy in most of our processes is ATP. However, it also provides precursors for many other processes such as synthesis of amino acids, for example. Thus, it is essential to have a strict regulation of glycolysis, so that the cell can respond to different needs of ATP or other metabolites.

During his studies on fermentation of glucose by yeast, Louis Pasteur discovered that the rate and amount of glucose consumed was higher in anaerobic than in aerobic conditions! At first glance this may seem strange, because aerobic metabolism is normally associated with something more advantageous for the cell. In fact, the biochemical explanation is simple: under anaerobic conditions one molecule of glucose generates 2 molecules of ATP, but under aerobic conditions generates 30 or 32 molecules of ATP. Simplifying, if one thinks that in five minutes the yeast will need to get 30 ATP, this means that under aerobic conditions it will only need to spend a molecule of glucose in that time, while under anaerobic conditions, as the process is less profitable in the energy point of view, it is necessary to spend 15 molecules of glucose. That is, the flow of glucose through the glycolytic pathway is regulated depending on cell ATP levels (as well as adequate supplies of glycolytic intermediates to biosynthetic roles).

As I have mentioned in previous posts, glycolysis has 10 reactions, and there are three regulatory points (irreversible reactions). The enzymes that catalyze them are the hexokinase (reaction 1), PFK-1 (reaction 3) and pyruvate kinase (reaction 10). As these reactions are the limiting steps of glycolysis, changes in speed of action of their enzymes will alter the overall speed of the glycolytic pathway.

Of the three regulatory enzymes, the main one is PFK-1. This may seem strange, because indeed the most logical situation was that the main regulatory enzyme was the first ... Again, there is a very simple explanation for this. What is happening is that hexokinase is an enzyme also common to other metabolic processes (synthesis of glycogen and pentose phosphate pathway). In other words, despite being a regulatory enzyme, it is not unique to glycolysis. Thus, the main point of regulation of glycolysis has to be the second, ie, the reaction catalyzed by PFK-1.

One thing I usually tell to my students is that in this part of metabolic regulation is preferable to understand why certain molecules activate and inhibit some pathways, thus avoiding to memorize endless lists of modulators... Of course it is not always possible to make a direct argument for understand why some molecules act as activators and others as inhibitors, but whenever possible I will develop this idea... Let's move on to a list of the main modulators of glycolysis.

Activators of hexokinase:
- Fructose-1-phosphate (liver) – It competes with fructose-6-phosphate to the regulatory protein of glucocinase, canceling its inhibitory effect.
- Inorganic phosphate (Pi) – It is a player in the glycolytic process (involved in reaction 6) so it makes sense that if it has a regulatory role, is a stimulating one.
Inhibitors of hexokinase:
- Glucose-6-phosphate (muscle) - It makes sense that functions as an inhibitor because it is the reaction product. If we have too much product, we will not need to continue to produce more ...
- Fructose-6-phosphate (liver) – It is the product of the following reaction (reaction 2), but can be interpreted the same way as the molecule before. That is, if we are to accumulate the intermediate formed from the reaction product, there is no point in continuing to make more product. This inhibition occurs through a protein called regulator protein of glucocinase.

Activators of PFK-1:
- Fructose-2,6-bisphosphate (liver) – It is the most significant allosteric regulator of PFK-1, reducing its affinity for the inhibitors ATP and citrate. It is produced in response to insulin and degraded in response to glucagon.
- Fructose-6-phosphate – It is the substrate, so it makes sense that if we have much substrate the enzyme is activated.
- ADP and AMP – They are produced when ATP is spent, thus indicating a low energy state. Therefore, it makes perfect sense that they can activate glycolysis, so that the cell can replenish their normal energy values. They activate the enzyme because they relieve the inhibition caused by ATP.Inhibitors of PFK-1:
- Glucagon (liver) - This hormone is produced in a state of hypoglycemia and aims to raise the concentration of glucose in the blood. So it makes perfect sense that it inhibits glycolysis, because this process consumes glucose, which will further accentuate the reduced blood glucose concentration. As mentioned earlier, the glucagon decreases the levels of fructose-2,6-bisphosphate
- ATP - The main objective of glycolysis is to produce energy (ATP). So if the cell already has ATP, it makes sense that glycolysis is inhibited, thus preventing an unnecessary waste of a precious metabolic fuel as glucose! ATP inhibits PFK-1 because it decreases the affinity of the enzyme for its substrate, fructose-6-phosphate.
- Citrate – It stresses the inhibitory effect of ATP. This molecule is the first intermediate of the following step of aerobic catabolism, the Krebs cycle. So if we are accumulating Krebs cycle intermediates, it is useless to continue to perform glycolysis.
- Phosphoenolpyruvate – It is an intermediate of glycolysis that is formed in the penultimate reaction. If there is an accumulation of this intermediate, the reactions above have to be inhibited in order to prevent a further accumulation of the molecule.
- H⁺ - This enzyme is particularly sensitive to changes in pH, functioning as a "switch" that turns off, for example, when we make an exaggerated lactic fermentation (produces H⁺), preventing an even greater acidification.

Activators of pyruvate kinase:
- ADP - The reason is the same as mentioned above for the PFK-1, ie, is an indicator of an energy deficit, so it will lead to an activation of glycolysis.
- Fructose 1,6-bisphosphate - an intermediate of glycolysis that is formed in a reaction prior to the one catalyzed by pyruvate kinase. So if we are accumulating an intermediate produced in an earlier stage, we have to activate this enzyme in order to counteract this accumulation (as when a dam is accumulating too much water, and to restore normal values ​​is necessary to open the gate ...).
- Dephosphorylation (liver) - Induced, for example, by insulin, which makes sense, given that insulin is produced in a situation of excess blood sugar (hyperglycemia) and will activate the process (one of them is glycolysis!) that consume glucose in order to lower the blood glucose concentration.
Inhibitors of pyruvate kinase:
- ATP – It is a carrier of chemical energy and one of the end products of glycolysis, so there is no need to continue the breakdown of glucose. It decreases the affinity of the enzyme for phosphoenolpyruvate.
- Acetyl-CoA – It is the molecule in which the product of this reaction (pyruvate) is converted in the case of aerobic catabolism. Therefore, if acetyl-CoA accumulates, it makes no sense to continue to synthesize pyruvate, so the enzyme is inhibited.
- Long-chain fatty acids.
- Phosphorylation (liver) - Induced, for example, by the action of glucagon, which, as mentioned earlier, will have as main function to raise blood glucose levels. To this end, it inhibits, for example, glycolysis.
- NADH - as we shall see in more detail when I speak of cellular respiration, NADH has potential to create molecules of ATP, which signals a high energy state of the cell. In this situation, it is not necessary to resort to glycolysis for more energy.
- Alanine - This amino acid (one of the 20 standard amino acids) can lead to pyruvate (the reaction product of pyruvate kinase!), by removal of its amino group. So if there is a molecule that can directly lead to pyruvate, we do not need to spend more glucose.

In short, we can make some generalizations about the regulation of metabolic pathways, which will be useful to understand the regulation of other processes.
First, energy molecules such as ATP, or potential energy, such as NADH are, in general, inhibitors of catabolism. This is very easy to understand if we think that the main objective of the catabolism is to produce energy. If the cell already has this ability it does not need to degrade more nutrients to produce energy! The oppposite reasoning applies to ADP, AMP and NAD⁺, because any one of these molecules indicates an energy deficit in the cell (remember that when we spend ATP we obtain ADP or AMP, and when we spend NADH we obtain NAD⁺...) so it will be necessary to restore energy levels, and this activates the catabolism.
Second, the reaction product or intermediates formed from this (products of reactions following the reaction we are considering) are inhibitors. On the other hand, the substrate, or intermediaries which will originate the substrate (formed in reactions prior to the reaction that we are considering) are activators.

Main bibliographic sources:

- Quintas A, Freire AP, Halpern MJ, Bioquímica - Organização Molecular da Vida, Lidel

- Nelson DL, Cox MM, Lehninger - Principles of Biochemistry, WH Freeman Publishers

Regulation of metabolism

Our body has a remarkable metabolic flexibility! Consider, for example, that we can adapt to situations as opposed as being 8-9 hours without eating (when you sleep, for example), or eat a very caloric meal.

Or else do a very intense workout in a short space of time, or a more moderate and longer, or simply stay at home. This ability to properly handle these opposites is a consequence of a strict regulation of our metabolic pathways.

The regulation of metabolic processes is, in my opinion the main point for a correct understanding of metabolism.

Before I start talking about specific regulation of each pathway, it is important to address some more general concepts ...
First, what is the regulation of metabolic pathways? It is the process by which the overall speed of each process is changed. Please note that when it comes to regulation, it does not mean necessarily inhibition, because the metabolic pathways can be activated or inhibited.
All pathways have at least one specific reaction of this process, which is irreversible. This ensures the cell two important ways:
1. Causes metabolic pathways do not occur in both directions, as a consequence only of the mass flow. That is, if a pathway produces the molecule X and the cell needs to produce more X, it is not because there is already this molecule within the cell that the same will be degraded.
2. They are used to regulate a specific metabolic pathway without affecting other processes, namely, the opposite process. To understand this we can think of two opposing processes, glycolysis (breakdown of glucose) and gluconeogenesis (glucose synthesis), for example. In cells the two processes do not occur simultaneously because there was no point being to degrade and synthesize glucose at the same time. Therefore, when one is active, the other must be inhibited. If both were catalyzed by the same enzymes, it was impossible to activate a process and inhibit another. Or both were activated or inhibited... How do we get around this problem? Using at least one enzyme specific for each process! So if I have a specific enzyme in glycolysis (there are 3 in fact ...) that does not act in gluconeogenesis, I can activate or inhibit this process without affecting the opposite. J

It is these very specific and irreversible reactions that are catalyzed by enzymes called regulatory. The regulatory enzymes are enzymes that act as a kind of metabolic pathways valves, allowing "flow" more intermediaries, if you lack more product, or accumulate these intermediates, if there is enough product. The reactions catalyzed by these enzymes are often referred to as points of regulation, considering that they are the limiting steps (slower) of the process to which they belong. So if their speed is increased, the overall speed of the pathway where they are located increases, and if its speed is decreased, the overall speed of the process decreases.

There are four types of regulation of metabolic pathways:
1. Substrate availability - is the fastest method of regulation and affects all the enzymes in each pathway. Basically, if there is few substrate, the enzymes will not be able to act at its maximum speed, and if there is no substrate, the enzymes stop.
2. Allosteric regulation - is the fastest way to regulate only certain specific enzymes, called regulatory enzymes. This form of regulation requires the presence of molecules (allosteric modulators) that will interact with the enzymes, leading to structural changes that can make the enzyme faster or slower (positive and negative modulators, respectively).

3. Hormonal regulation - is a slower process than the allosteric regulation, and involves the production of hormones in response to a stimulus. Hormones are released into the bloodstream and will act on target cells. Usually its action culminates in the phosphorylation or dephosphorylation of regulatory enzymes, altering their catalytic efficiency (activated or inhibited, depending on the enzyme in question). This effect is called reversible covalent modification.

4. Changes in the concentration of enzymes - This is the slowest way of regulation and include changes in rates of synthesis and degradation of enzymes, altering their concentration. For example, if a cell wants to activate a metabolic pathway, it can do so by increasing the amount of the enzymes of this pathway. Since the substrate is not limiting, the overall rate of conversion of substrate to product will increase. The opposite effect is observed by the opposite reasoning.

Main bibliographic sources:

- Quintas A, Freire AP, Halpern MJ, Bioquímica - Organização Molecular da Vida, Lidel

- Nelson DL, Cox MM, Lehninger - Principles of Biochemistry, WH Freeman Publishers