Krebs cycle what is produced

If ATP levels increase, the rate of this reaction decreases. If ATP is in short supply, the rate increases. Step 2. Citrate loses one water molecule and gains another as citrate is converted into its isomer, isocitrate. Steps 3 and 4. CoA binds the succinyl group to form succinyl CoA.

Step 5. A phosphate group is substituted for coenzyme A, and a high- energy bond is formed. This energy is used in substrate-level phosphorylation during the conversion of the succinyl group to succinate to form either guanine triphosphate GTP or ATP.

There are two forms of the enzyme, called isoenzymes, for this step, depending upon the type of animal tissue in which they are found. One form is found in tissues that use large amounts of ATP, such as heart and skeletal muscle.

This form produces ATP. The second form of the enzyme is found in tissues that have a high number of anabolic pathways, such as liver.

This form produces GTP. In particular, protein synthesis primarily uses GTP. Step 6. Step six is a dehydration process that converts succinate into fumarate. Unlike NADH, this carrier remains attached to the enzyme and transfers the electrons to the electron transport chain directly.

This process is made possible by the localization of the enzyme catalyzing this step inside the inner membrane of the mitochondrion. Step 7. Water is added to fumarate during step seven, and malate is produced. The last step in the citric acid cycle regenerates oxaloacetate by oxidizing malate. Another molecule of NADH is produced. Two carbon atoms come into the citric acid cycle from each acetyl group, representing four out of the six carbons of one glucose molecule. Two carbon dioxide molecules are released on each turn of the cycle; however, these do not necessarily contain the most recently-added carbon atoms.

The two acetyl carbon atoms will eventually be released on later turns of the cycle; thus, all six carbon atoms from the original glucose molecule are eventually incorporated into carbon dioxide. These carriers will connect with the last portion of aerobic respiration to produce ATP molecules. Several of the intermediate compounds in the citric acid cycle can be used in synthesizing non-essential amino acids; therefore, the cycle is amphibolic both catabolic and anabolic. ATP-citrate lyase links metabolism to histone acetylation as it converts glucose-derived citrate to acetyl-CoA and it has been found to localize to both nucleus and cytoplasm Citrate is small enough to diffuse across nuclear pores allowing for acetyl-CoA to be produced in either cellular compartment, and siRNA-mediated knockdown of ACLY reduced global histone acetylation Since ACLY is upregulated in lipopolysaccharide LPS -stimulated macrophages 49 , it would be interesting to see where ACLY localized to and if there was a direct effect on the expression of glycolytic genes due to changes in histone acetylation.

ACLY has been shown to control glucose to acetate switch. ACLY-deficient cells upregulate ACSS2 allowing for the production of acetyl-CoA from acetate, ensuring cell viability and providing substrates for both fatty acid synthesis and histone acetylation As previously discussed, in glucose-deprived conditions, increased flux through CIC can sustain NADPH levels in glucose-deprived activated macrophages Acetylation of CIC increases in glucose-deprived growth conditions compared to media containing glucose.

By reconstituting liposomes with mitochondrial extracts, it was shown that the acetylation of CIC causes an increase in V max for citrate Acetate is taken up and processed via the Krebs cycle to produce citrate. Though metabolic reprogramming differs in activated T cells compared to macrophages and DCs, this highlights the importance of the citrate pathway in control of both the metabolism of immune cells and their production of pro-inflammatory mediators, unlike in T cells.

No work has yet been carried out to directly link citrate-derived acetylation in M1 macrophages or DCs, however, histone acetylation is important in macrophage activation and DC differentiation. IL-6 and IL production are both regulated on histone and non-histone protein acetylation, respectively 78 , Therefore, it is likely that acetylation plays a role in the regulation of immune cell metabolism. Histone acetylation downstream of ACLY has been shown to be of importance in M2 macrophage activation While STAT6 is the major regulator of IL4 induced genes a subset of genes important in the regulation of cellular proliferation and the production of chemokines are under additional control of an Akt—mTORC1 signaling pathway.

Covarrubias et al. They suggest that certain transcription factors and histone acetyltransferases, e. Malonyl-CoA is the cofactor required Lysine-malonylation has been shown to play a role in the regulation of mitochondrial function, FAO and glycolysis 86 , Notably histone malonylation does not occur at the N-terminal tail as happens with acetylation, suggesting that the regulatory role these two modification carry out may be very functionally different to acetylation A large number of proteins involved in fatty acid metabolism are malonylated, including ACLY.

However, no studies have yet been carried out regarding the functional consequence of lysine-malonylation in immune cells.

While the accumulation of citrate caused by the IDH1 breakpoint in the TCA cycle can be used to fuel fatty acid synthesis and histone acetylation, another fate of this citrate is the production of itaconate Figure 3. First identified in as a product of the distillation of citric acid, itaconate has recently become a focus of the field of immunometabolism due to its potential role as an anti-inflammatory modulator.

Itaconate is derived from citrate produced in the Krebs cycle and, in M1 macrophages, is one of the most highly induced metabolites following LPS treatment Citrate is acted on by the mitochondrial aconitase 2 ACO2 to produce cis-aconitate. Cis-aconitate is decarboxlylated by cis-aconitate decarboxylase, also known as immune-responsive gene 1 IRG1 , to produce itaconate.

Itaconate has long been used in an industrial setting and is produced on an industrial scale as a fermentation product of Aspergillus terreus for use in the creation of polymer formation Figure 3. Citrate-derived itaconate. Citrate is converted to cis-aconitate by the Krebs cycle enzyme ACO2. Activation of macrophages with LPS causes induction of IRG1 which can produce itaconate by the decarboxylation of cis-aconitate. Itaconate is toxic to microorganisms expressing ICL, a key component of the glycoxylate shunt in bacteria.

In , it was found that immunoresponsive gene 1 Irg1 is highly upregulated in peritoneal macrophages following LPS stimulation 89 , and has since been seen to be upregulated in the blood of human sepsis patients 90 and in the time during embryo implantation 91 , Despite lacking a sequence targeting it, there IRG1 has been found to associate with the mitochondria 93 , It was only in that itaconate was identified in multiple studies in an immune context and in that IRG1 and itaconate were connected Itaconate was seen in the lungs of mice infected with Mycobacterium tuberculosis MTB and was not present in the lungs of control mice In a separate study, itaconate was shown to be secreted by the macrophage cell line RAW Michelucci et al.

They further showed by isotope-labeling that itaconate was derived from citrate. Genetic silencing of Irg1 causes macrophages to lose their bactericidal activity, which was due to decreased amounts of itaconate and the loss of its inhibitory effect on isocitrate lyase ICL , a crucial enzyme of the glycoxylate shunt in bacteria The glycoxylate shunt is a means for bacteria to survive in conditions of low glucose availability where acetate is the primary fuel source.

Succinate enters the Krebs cycle and glycoxylate is then converted to malate by malate synthase. Malate can be processed to oxaloacetate by MDH as in the normal reactions of the Krebs cycle. Some bacteria are able to degrade itaconate, producing acetyl-CoA and pyruvate, due to the expression of genes that encode for itaconate-CoA transferase, itaconyl-CoA hydratase, and S -citramalyl-CoA ligase.

Possession of these genes allows Pseudomonas aeruginosa and Yersinia pestis to survive in activated macrophages There is a discussion as to the relevance of these studies due to differences in concentrations of itaconate used both in terms of the variety of concentrations used exogenously to inhibit bacterial growth and the range in reported intracellular concentrations , It may be that intracellular itaconate is concentrated in vacuoles, and whole cell analysis will not adequately represent this, and that measuring the concentration of secreted itaconate in cell culture media does not determine what the local concentration would be.

While the effect of itaconate on bacterial survival has been well documented, more recent work has sought to elucidate the effect that a high intracellular concentration of itaconate has on the immune cells that produce it. Dimethyl itaconate DMI has been used in several studies as a cell permeable itaconate analog to boost the intracellular levels of itaconate.

The authors suggest that this is due to the ability of itaconate to inhibit SDH, and they showed itaconate to inhibit a purified form of SDH. As SDH also acts as complex II of the ETC, this highlights the ability of endogenous itaconate to regulate mitochondrial metabolism and is consistent with other reports of itaconate competitively inhibiting SDH, albeit weakly, the first of which was in — This led Lampropoulou et al.

When succinate accumulates and is oxidized by SDH, it will produce a large amount of coenzyme Q. IRG1 has also been shown to play a role in the establishment of endotoxin tolerance in LPS-tolerized macrophages. Several other elements regulating IRG1 expression and, therefore, itaconate production have also recently been identified. Inhibition of branched-chain aminotransferase 1 in human monocyte-derived macrophages decreased levels of glycolysis and oxygen consumption while also reduced IRG1 mRNA and protein levels as well as itaconate production A major issue with the study of the functional effect of itaconate in macrophages to date has been the use of DMI.

DMI was utilized as it is cell permeable, however, it has been shown that while DMI boosts the level of itaconate in the cell it is not itself metabolized to itaconate El Azzouny et al. The authors speculate that the effects of DMI on macrophage metabolism may be due to an ability to act as a cysteine alkylating agent or to alter redox homeostasis. They further suggest that, though one has not been identified, it is possible a cell surface receptor for itaconate exists that DMI would be able to bind.

While the effects of studies carried out utilizing DMI have been drawn into question, the body of work carried out using genetic inhibition or deletion of Irg1 and the striking amount by which Irg1 mRNA and itaconate synthesis are upregulated in activated immune cells still leaves it worthy of further investigation. Our understanding of immune cell metabolism has come far since the early observations that activated macrophages were highly glycolytic , It is now well accepted that these pathways play a part outside of their traditional energetic and biosynthetic roles.

The discovery that the Krebs cycle is not complete in activated M1 macrophages and DCs highlights the importance of the withdrawal of citrate from the cycle for DC activation, the production of pro-inflammatory mediators and for the generation of itaconate. Citrate links many important cellular processes, bridging carbohydrate and fatty acid metabolism and protein modification. Its role in producing acetyl-CoA for the acetylation of histones may turn out to be its most striking role in regulating immune cell function.

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Science — The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature —5. J Clin Invest 10 — HIFmediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab 3 3 — But really, for the steps after glycolysis you get rid of three carbons. But you're going to do it for each of the pyruvates. You're going to get rid of all six carbons, which will have to exhale eventually.

But this cycle, it doesn't just generate carbons. So we'll write that here. And this is a huge simplification. I'll show you the detailed picture in a second. We'll do it again. And of course, these are in separate steps. There's intermediate compounds. I'll show you those in a second. It will produce some ATP. And the whole reason why we even pay attention to these, you might think, hey cellular respiration is all about ATP.

The reason why we care is that these are the inputs into the electron transport chain. These get oxidized, or they lose their hydrogens in the electron transport chain, and that's where the bulk of the ATP is actually produced.

And then maybe we'll have another NAD get reduced, or gain in hydrogen. Reduction is gaining an electron. Or gaining a hydrogen whose electron you can hog. And then we end up back at oxaloacetic acid. And we can perform the whole citric acid cycle over again. So now that we've written it all out, let's account for what we have. So depending on-- let me draw some dividing lines so we know what's what.

So this right here, everything to the left of that line right there is glycolysis. We learned that already. And then most-- especially introductory-- textbooks will give the Krebs cycle credit for this pyruvate oxidation, but that's really a preparatory stage.

The Krebs cycle is really formally this part where you start with acetyl-CoA, you merge it with oxaloacetic acid. And then you go and you form citric acid, which essentially gets oxidized and produces all of these things that will need to either directly produce ATP or will do it indirectly in the electron transport chain.

But let's account for everything that we have. Let's account for everything that we have so far. We already accounted for the glycolysis right there.

Now, in the citric acid cycle, or in the Krebs cycle, well first we have our pyruvate oxidation. That produced one NADH.

But remember, if we want to say, what are we producing for every glucose? This is what we produced for each of the pyruvates. This NADH was from just this pyruvate. But glycolysis produced two pyruvates. So everything after this, we're going to multiply by two for every molecule of glucose. And then when we look at this side, the formal Krebs cycle, what do we get?

We have, how many NADHs? One, two, three NADHs. So three NADHs times two, because we're going to perform this cycle for each of the pyruvates produced from glycolysis. So that gives us six NADHs. We have one ATP per turn of the cycle. That's going to happen twice. Once for each pyruvic acid. So we get two ATPs. And then we have one FADH2. But it's good, we're going to do this cycle twice. This is per cycle. So times two. We have two FADHs.

So sometimes instead of having this intermediate step, they'll just write four NADHs right here. And you'll do it twice. Once for each puruvate. But the reality is, six from the Krebs cycle two from the preparatory stage. Now the interesting thing is we can account whether we get to the 38 ATPs promised by cellular respiration. So we have four ATPs. Four ATPs. How many NADHs do we have?

We have 10 NADHs. And then we have 2 FADH2s. It should be FADH2, just to be particular about things. And these, so you might say, hey, where are our 38 ATPs? We only have four ATPs right now. But these are actually the inputs in the electron transport chain. These molecules right here get oxidized in the electron transport chain. So two of them are going to produce four ATPs in the electron transport chain. So we now see, we get four from just what we've done so far.

Glycolysis, the preparatory stage and the Krebs or citric acid cycle. And then eventually, these outputs from glycolysis and the citric acid cycle, when they get into the electron transport chain, are going to produce another So 34 plus 4, it does get us to the promised 38 ATP that you would expect in a super-efficient cell.

This is kind of your theoretical maximum. In most cells they really don't get quite there. But this is a good number to know if you're going to take the AP bio test or in most introductory biology courses.

There's one other point I want to make here. Everything we've talked about so far, this is carbohydrate metabolism. Or sugar catabolism, we could call it. We're breaking down sugars to produce ATP. Glucose was our starting point. But animals, including us, we can catabolize other things. We can catabolize proteins. We can catabolize fats. If you have any fat on your body, you have energy.