Cartoon - Krebs cycle

Thank you for the input Ferraz! :)
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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.

Krebs cycle (enzymes) - Part 2

After a “troubled” end of school year (as always ...) and a vacation offline period, I am back to the posts. :)
In this post I will continue to describe the main characteristics of the Krebs cycle enzymes...

Succinyl-CoA synthetase
This enzyme, also called tiocinase succinate or succinate-CoA ligase, presents in its composition two subunits (alpha and beta). The alpha subunit binds to the CoA molecule, while the beta subunit binds GDP. There is an isoform of the enzyme (also mitochondrial) which has beta subunit with affinity to ADP, instead of GTP.

 

The mechanism of reaction occurs in three steps. The succinyl-CoA synthetase has a histidine residue which plays a central role in the transfer of the phosphate group to the biphosphate nucleotide that is bound to the beta subunit.

Failures in succinyl-CoA synthetase are the cause of the disease "fatal child lactic acidosis," which is a disease characterized by the production of high levels of lactic acid (which is easily understanded because of the Krebs cycle is a step of carbohydrates aerobic catabolism), which would normally cause death of the individual within the first 4 days of life.

Succinate dehydrogenase
This enzyme, also called succinate-coenzyme Q reductase, belongs simultaneously to the Krebs cycle and the mitochondrial respiratory chain, where is known as complex II. Because of this, it is the only Krebs cycle enzyme that is associated with the inner mitochondrial membrane (all the others are present in the matrix ...). It uses as a cofactor FAD.

Structurally, it presents four subunits, two hydrophobic and two hydrophilic ones. The first two are a flavoprotein (SdhA) and an iron-sulfur protein (SdhB). SdhA is the subunit that binds covalently FAD and succinate, while SdhB is characterized by the presence of three iron-sulfur clusters ([2Fe-2S], [4Fe-4S] and [4S-3Fe]). The hydrophobic subunits (SdhC and SdhD) function as membrane anchors. The two hydrophobic subunits form the cytochrome b, characterized by having six transmembrane domains, a heme group and a binding site for ubiquinone (which also involves subunit SDHB).

The binding site for succinate (subunit SdhA) involves the side chains of important amino acid residues, in particular Threonine254, Histidine354 and Arginine399.
The binding site for ubiquinone requires the presence of some essential amino acid residues, namely Proline160, Tryptophan 163, Tryptophan164, Histidina207 and Isoleucine209 (subunit B), Serine27, Isoleucine28 and Arginine31 (subunit C) and Tyrosine83 (subunit D).
Failures in the succinate dehydrogenase can lead to the appearance of several pathologies, including:
- Leigh syndrome, mitochondrial encephalopathy and optic atrophy (mutations in SdhA).
- Hereditary paraganglioma, hereditary pheochromocytoma and excessive production of superoxide ions (mutations in SdhB, SdhC and/or SdhD).

Fumarase
This enzyme, also known as fumarate hydratase or malate hydrolyase, has two isoforms, one mitochondrial and other cytosolic. It is a tetrameric enzyme, and the substrate binding site is called the catalytic center A and involves amino acid residues from three different subunits.

The enzyme is present in two forms, E1 and E2. The first is characterized by two acid/base groups (essential for its catalytic activity) without charge, being responsible for binding to the fumarate and subsequent chemical transformation in malate. The form E2 has the two acid/base groups in the ionized form of zwitterion (one with positive charge and one with negative charge), characterized by binding to malate. Both forms are interconverted during the catalytic cycle of the enzyme.

Deficiency in fumarase is called polyhydramnios and is also associated with the appearance of skin and uterus leiofibromyomas and renal carcinoma.

Malate dehydrogenase
The malate dehydrogenase has two distinct isoforms, a mitochondrial one (isoform 2) and other cytosolic (isoform 1). It is an enzyme which not only plays a role in Krebs cycle, but it is also involved in gluconeogenesis.

Structurally, it has similarities to lactate dehydrogenase, with a homodimeric structure (subunits with masses of 30-35 kDa). Each subunit has two domains, the first of which is characterized by a beta-sheet structure, while the other represents the binding site to NAD+, composed of four beta-sheet and one alpha helix. The subunits interact with one another through hydrogen bonding and hydrophobic interactions.
The active site of the enzyme is essentially hydrophobic, with separate binding sites for malate and NAD+. It presents some particular amino acid residues important for its catalytic activity, namely the Arginine102, Arginine109, Aspartate168, Arginine171 and Hystidine195.

Music about citrate

Here it is a music made by Dr. Ahern (www.davincipress.com/metabmelodies.html), this time about the firts intermediate of Krebs cycle, the citrate. It was inspired in the song God Rest Ye Merry Gentlemen.

Citrate Sonata


Our fats and carbs get broken down
To acetyl CoA
Oxaloacetate combines
In cycles TCA
The product of reaction one
Oh, citrate is its name
Iso-citrate, the product that ensues
Atoms got moved
Isocitrate is the product of step two

An oxidation soon occurs
Reducing NAD
An alpha-ketoglutarate
Resulting from step three
From here we could make glutamate
That is, if there’s a need
Don’t forget that we lost a CO2
Yes it is true
In reaction three we lost a CO2

So what’s the point of all these steps?
Well, let me tell you, friend
We use electron carriers
In working towards our end
Of synthesizing ATP
(A metabolic trend)
Oxidize, and then oxidize some more
Here in step four
Ketoglutarate gets oxidized some more

The enzyme with cofactors five
Including TPP
Lipoate, FAD, CoA
And also NAD
A succinyl that’s on CoA
Is what gets made, you see
This reaction occurs so fav’rably
Don’t you agree?
It’s a good reaction energetically

With four more steps, we’re halfway there
So let me summarize
When CoA’s lost we see that GT-
P is synthesized
The succinate that is produced
Will soon get oxidized
FAD goes to FADH2
What did we do?
We made fumarate and FADH2

Add water ‘cross the double bond
And malate we create
With one last NAD we can
Then dehydrogenate
To give a final product of
Oxaloacetate
It’s removed, and this lowers Delta G
Oh yes, indeed
It’s through pulling that this last step can proceed
.

Krebs cycle (enzymes) - part 1

The Krebs cycle is a metabolic pathway composed of 8 biochemical reactions, each catalyzed by a different enzyme. Here is some information about the first four enzymes of the Krebs cycle ...

Citrate synthase

The citrate synthase is an enzyme widely used as a biomarker for the presence of intact mitochondria in cell cultures or organelle preparations. Despite being a mitochondrial enzyme it is encoded by nuclear DNA and synthesized in the cytosol.

This enzyme is the first regulatory enzyme in the Krebs cycle. It uses two different substrates, the acetyl-CoA and oxaloacetate. The oxaloacetate firstly binds to the enzyme, which induces conformational changes that create the binding site for the acetyl-CoA molecule. 

From a structural point of view, it is composed of 437 amino acid residues and has two subunits, each with about 20 alpha helices. The active center has three amino acid residues essential for the catalytic function of the enzyme, due to the establishment of specific interactions with the substrates - His274, His320, and Asp-375.

 

Its mechanism of action involves an aldol condensation. To view a video about the mechanism of action of citrate synthase, click here.

Aconitase

The aconitase is an enzyme that has a functional iron-sulfur cluster [Fe₄S₄]²⁺, which interacts with three cysteine ​​residues of the enzyme. It is especially sensitive to oxidative stress and, in particular, to superoxide anion, due to the iron-sulfur cluster. 

It has two homologues in our body, the iron-responsive element-binding protein (IRE-BP) and the 2-isopropylmalate dehydratase (or alpha-isopropylmalate isomerase).

From a structural standpoint, the aconitase has two conformations, one for the inactive and one for the active state. In the inactive form, it has four domains, the first three establish interactions with the iron-sulfur cluster, while the latter has the active center. When it becomes active, the enzyme is altered in the iron-sulfur cluster (Fe₃S₄ turns in Fe₄S₄), and this represents the main difference between the two conformations of the enzyme.

Its mechanism of action relies on a mechanism of dehydration-hydration, via the intermediate cis-aconitate.

Its active site has two amino acid residues particularly important for catalytic activity - His101 and Ser642.

The importance of this enzyme, in a physiological point of view, is supported by the existence of many diseases that affect it. One is referred to as aconitase deficiency. It is caused by a mutation in the gene that codes for a protein responsible for the assembling of the iron-sulfur cluster. This disease causes myopathy and exercise intolerance, because the aerobic catabolism of these individuals is compromised. Another disease is Friedreich's ataxia (FRDA), characterized by a lower activity of aconitase and other Krebs cycle enzyme, the succinate dehydrogenase. Besides these, there are studies that suggest a possible relationship between aconitase and diabetes. However, it is still an hypothesis that has to be best characterized.

 
Isocitrate dehydrogenase

Isocitrate dehydrogenase is the second regulatory enzyme in the Krebs cycle. There are three different isoforms of isocitrate dehydrogenase. One exists only in the mitochondrial matrix and uses NAD⁺ as the acceptor of electrons. The other isoforms use NADP⁺ as the acceptor of electrons and appear to have as main function the formation of NADPH, essential for the reducing anabolic reactions. These forms are present in the mitochondrial matrix, the cytosol and in the peroxisome. 

The forms using NADP⁺ as a cofactor have an homodimeric structure, while the one that uses NAD⁺ is a heterotetramer.

The reaction catalyzed by isocitrate dehydrogenase involves the formation of an intermediary, the oxalossuccinate.

From the clinical point of view, some mutations were found in isocitrate dehydrogenase in some brain tumors, including astrocytoma, oligodendroglioma and multiforme glioblastoma. There are also some studies that indicate a possible relationship between mutations in the enzyme and acute myeloid leukemia.

Alpha-ketoglutarate dehydrogenase

This is the third (and last!) regulatory point of the Krebs cycle.

This enzyme, which can also be referred to as oxoglutarate dehydrogenase, is actually a multienzyme complex. It consists of the following enzymes: alpha-ketoglutarate dehydrogenase, dihydrolipoyl succinyltransferase dihydrolipoyl dehydrogenase. It has a structure and a reaction mechanism very similar to the pyruvate dehydrogenase complex. Because of this, it is believed that possibly both complexes had a common origin and at some point of evolution they suffered a divergent evolution.

Clinically, this enzyme complex functions as an autoantigen in primary biliary cirrhosis, a form of acute hepatic failure. Moreover, its catalytic activity is also decreased in various neurodegenerative diseases.

Video about Krebs cycle (4)

Video about the Krebs cycle (3)

This video is different... it is a Legos's version of the Krebs cycle! :)

Krebs cycle (reactions) - part 2

Reaction 5: conversion of succinyl-CoA to succinate This reaction requires Mg2+. The enzyme that catalyzes this reaction, succinyl-CoA synthetase, breaks the thioester bond (S-CoA), releasing a large amount of energy that is used to phosphorylate GDP to GTP. It is another example of an energy coupling.

Reaction 6: oxidation of succinate to fumarate

Succinate is oxidized to fumarate, leading to the production of FADH2 from FAD. The reaction is catalyzed by succinate dehydrogenase, which is the only Krebs cycle enzyme that is not present in the matrix, but instead is strongly associated with the inner membrane of mitochondria.

Step 7: Hydration of fumarate to malate
This enzyme is highly stereo-specific, producing only the stereoisomer L-malate. The reaction is reversible in cellular conditions.

Step 8: oxidation of malate to oxaloacetate
This reaction produces a molecule of NADH from NAD+. At the intracellular conditions, the reaction is mainly driven in the opposite direction, but as the oxaloacetate is continuously removed (by the reaction of synthesis of citrate, by gluconeogenesis or by transamination to originate aspartate), the equilibrium is shifted in the forward direction. The oxaloacetate used in the first reaction of the Krebs cycle is then regenerated, so, theoretically, one molecule of oxaloacetate may be involved in the oxidation of an infinite number of molecules of acetyl-CoA (playing a kind of "catalytic" role) and, therefore, the oxaloacetate is present in cells at very low concentrations.

  

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

Video about the Krebs cycle (2)

Here it goes one more video about the Krebs cycle, this time in a karaoke version. :)

Krebs cycle (reactions) - part 1

Reaction 1: formation of citrate
This irreversible reaction is the 1st regulatory point of the Krebs cycle. It is a reaction in which an oxaloacetate molecule reacts with acetyl-CoA. In this process it is formed a very energetic intermediate (citroil-CoA) that rapidly converts into citrate. The molecule of CoA-SH liberated is recycled to participate in a new oxidative decarboxylation of pyruvate (catalyzed by pyruvate dehydrogenase complex).

Reaction 2: formation of isocitrate via cis-aconitate

This reaction occurs through the formation of an intermediate, cis-aconitate, obtained by dehydration of citrate. Thereafter, the cis-aconitate is hydrated, forming isocitrate. Thus, citrate and isocitrate are isomers. Despite that in cellular conditions the reaction produces only about 10% of isocitrate, the rapid consumption of this product in the following reaction shifts the equilibrium in the forward direction. The fluoroacetate is a toxic molecule because in physiological conditions it is transformed into fluoroacetil-CoA, which condenses with oxaloacetate to form fluorocitrate, that inhibits aconitase, causing accumulation of citrate.

Reaction 3: oxidation of isocitrate to α-ketoglutarate and CO2This reaction is an example of an irreversible oxidative decarboxylation, and it is the 2nd regulatory point in the Krebs cycle. In fact, this reaction is a set of three different reactions:
1. Dehydrogenation of isocitrate, creating oxalosuccinate and producing NADH.
2. Binding of Mn2+ to the carbonyl group of oxalosuccinate, stabilizing the enol and promoting the release of CO2.
3. Hydrogenation, with the arrangement of the resonance hybrid.

Reaction 4: Oxidation of α-ketoglutarate to succinyl-CoA and CO2This reaction, like the previous one, is another example of an irreversible oxidative decarboxylation. It is the 3rd (and the last one!) regulatory point of the Krebs cycle. This reaction is virtually identical to the oxidative decarboxylation of pyruvate, also leading to the formation of NADH. It is a very exergonic reaction due to the energy stored in the bond S-CoA.

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

Krebs cycle (general ideas) - part 2

For each round of the Krebs cycle, three molecules of NADH, 1 FADH2, 1 ATP (GTP) are produced.

The passage of electrons from a molecule of NADH to O2 on oxidative phosphorylation leads to the formation of 2.5 molecules of ATP. If the donor electron is the FADH2 only 1.5 molecules of ATP are formed. Therefore, one molecule of glucose that is completely oxidized to CO2 via glycolysis, pyruvate dehydrogenase, Krebs cycle and oxidative phosphorylation, produces 32 ATP molecules.

The Krebs cycle plays a central role in cellular metabolism, because all the nutrients that may play a role "energy" generated in its catabolism acetyl-CoA.In addition to oxidize acetyl-CoA to CO2 and to produce ATP, NADH and FADH2, also receives several intermediaries arising from several catabolic pathways. Oxaloacetate and α-ketoglutarate, for example, are the products of decomposition of aspartate and glutamate. In addition to receiving several intermediates from catabolic processes, it also provides various intermediates for anabolic pathways. Because of this feature (involvement on both anabolic and catabolic processes) the Krebs cycle is an amphibolic process.

Oxaloacetate and α-ketoglutarate are also precursors of amino acids and purine and pyrimidine bases. Oxaloacetate is converted to glucose in gluconeogenesis, succinyl-CoA intermediate in the synthesis of the porphyrin ring of heme groups.

When the Krebs cycle intermediates are diverted to biosynthetic processes, their stock quantity is replenished by anaplerotic reactions. The most important reaction in the liver and kidneys is the reversible carboxylation of pyruvate to oxaloacetate. The enzyme that catalyzes this reaction is pyruvate carboxylase and is stimulated by acetyl-CoA. Another important reaction is the carboxylation of phosphoenolpyruvate to oxaloacetate. The enzyme that catalyzes this reaction is phosphoenolpyruvate and is stimulated by fructose-1,6-bisphosphate. Other important anaplerotic reactions are transaminations, to obtain amino acids (the intermediate of cycle provides the carbon skeleton). The cycle also provides intermediates in the synthesis of glucose (gluconeogenesis) and fatty acids.

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

Krebs cycle (general ideas) - part 1

The Krebs cycle is also called the citric acid cycle or the tricarboxylic acids cycle. It is a catabolic process that occurs in mitochondria, specifically in the mitochondrial matrix (as I will highlight in a next post, there is just a reaction that occurs in association with the inner membrane of mitochondria). In the cycle, cells oxidize acetyl-CoA molecules to CO2, and the energy released is conserved in the form of NADH and FADH2. The Krebs cycle is exclusively aerobic, because although O2 does not participate directly in the cycle, the NAD + and FAD can only be regenerated in the mitochondria by transferring electrons to O2 (in the post on the regulation of the Krebs cycle, which will place soon, it will be possible to see that if NADH accumulates, that is what happens in the absence of O2, the Krebs cycle is inhibited ...).
We oxidize in the Krebs cycle many moles of acetyl-CoA per day. The oxidants, NAD+ and FAD, are reduced to NADH and FADH2. In the cell there are only a few micromoles of NAD + and FAD and within the mitochondria (where the cycle occurs) the regeneration of NAD+ and FAD depends on the respiratory chain, so the Krebs cycle does not occur under anaerobic conditions. The Krebs cycle is like a "mill" where the "grain" (the substrate) is the acetyl group of acetyl-CoA and the "flour" (products) are CO2 and electrons (NADH and FADH2), the "millstone" are the enzymes and intermediate compounds.

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

Game about Krebs cycle

Here is the link of a game about Krebs cycle.

http://www.purposegames.com/game/0e38fb1b/info

Fates of pyruvate

The fate of pyruvate depends on cell type and metabolic conditions. There are three main destinations for pyruvate:

(1) aerobic organisms and tissues, under aerobic conditions - pyruvate is oxidized, with loss of the carboxylic group, resulting in the acetyl group from acetyl-CoA, which is then oxidized to CO₂ in the Krebs cycle;

(2) Aerobic tissues in conditions of low oxygen (muscle hypoxia, for example), some tissues under aerobic conditions (red cells, for example, because they lack mitochondria), or some anaerobic organisms - pyruvate is reduced to lactate by lactic fermentation . In muscle under conditions of hypoxia, NADH is not reoxidized to NAD⁺ and NAD⁺ is required for glycolysis. The reduction of pyruvate to lactate allows the use of as a donor of electrons to regenerate NAD⁺;

(3) Some tissues of plants, some invertebrates, protists and micro-organisms under anaerobic conditions or hypoxia - pyruvate is converted to ethanol + CO₂ (alcoholic fermentation).

While glycolysis can occur in anaerobic conditions, this fact has a price, because it reduces the amount of ATP formed per molecule of glucose (from 30 or 32 it passes for only 2 ATP!), and, therefore, it is needed more glucose oxidation under these conditions.

What happens to pyruvate is directly related to the amount of NAD⁺ and FAD in the cell. As these amounts are very small, there must be mechanisms to transform the NADH + H⁺ and FADH₂ back into NAD⁺ and FAD, respectively. This is done by transfer of the electrons from NADH + H⁺ and FADH₂ to other molecules, which can occur by fermentation or respiration. The distinction between these is not (contrary to what is generally thought) that one of the processes uses directly the O₂ and the other not! O₂ is only required for oxidative phosphorylation, and not for the oxidation of pyruvate. Unlike aerobic metabolism that depends and are limited by the oxygen supply, the anaerobic glycolysis does not depend on the availability of oxygen and can increase speed up to 1000 times the speed at rest, ie, 2 ATP / glucose can represent many ATP/minute.

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