---
title: The Citric Acid Cycle
disqus: hackmd
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The Citric Acid Cycle
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> Documentation [name=MrDr.Staffan]
###### tags: `page`, `metabolism`
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### Table of Contents
[TOC]
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[Top](#Table-of-Contents)

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A novel look at the TCA.

2021
Reverse TCA. How bacteria, with excess energy and CO2 form metabolites
https://www.nature.com/articles/d41586-021-00977-1

"Yet, surprisingly, H2 and CO2 can form pyruvate overnight without any enzyme involvement if simple metal catalysts, of the kinds found in the oceanic crust, are provided9. This suggests that early metabolism on Earth was built around the naturally occurring chemistry between CO2 and H2 in mineral-rich environments10."

---

[Also See Circuits - energetics](https://hackmd.io/@sholmqvist/BJpKurTBB/%2FKEQ0Opd0QhGGWxXP3NqVIA)

- Pyruvate dehydrogenase
- Pyruvate dehydrogenase is the enzyme that forms acetyl coenzyme A (acetyl CoA) from pyruvate.

- In the TCA cycle, the acetyl part of acetyl CoA, which contains two carbon atoms, condenses with a preexisting four-carbon TCA intermediate, oxaloacetate (OAA), to form a six-carbon TCA cycle constituent (citrate).

- Citrate via two decarboxylations (The TCA) regenerates OAA, ready to condense with another molecule of acetyl CoA.

- Carboxylation of pyruvate
- In addition to being regenerated in each turn of the TCA cycle, OAA can be formed by carboxylation of pyruvate
- an anaplerotic process (=forming intermediate), expanding the concentration of TCA cycle intermediates and catalyzed by the astrocyte-specific PC.
- This enzyme is virtually absent in neurons.

---

G6P // GAPD
This is not only a glycerol with a single phosphate. It is also the famous GAPD in GAPDH. Used for normalizations. That is normalizations against maintained rate of glycolysis+glyconeogenesis acitivty.

[](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6165802/)

# Common pathway for oxidation of fuels.

Under aerobic conditions, the pyruvate generated from glucose is oxidatively decarboxylated to form acetyl CoA.

# Anaplerotic
[Pyruvate absorbs CO2 and expands TCA forming Oxaloacetat. Needs biotin.]

[See Biotin](https://hackmd.io/@sholmqvist/BJpKurTBB/%2FVRxXizYGTSmR_hDbuSI8LQ#Vitamin-B7-Biotin)



# Malate synthase
[Very speculative, perhaps this is happening in astrocytes. or something similar. Astrocytes lack SDH//ComplexII gene expressions. Instead astrocytes have ]
Malate synthase is used by plants in the glyoxylate cycle to incorporate Acetate into the cycle.

The glyoxylate cycle, a variation of the tricarboxylic acid cycle, is an anabolic pathway occurring in plants, bacteria, protists, and fungi. The glyoxylate cycle centers on the conversion of acetyl-CoA to succinate for the synthesis of carbohydrates.[1] In microorganisms, the glyoxylate cycle allows cells to utilize two carbons (C2 compounds), such as acetate, to satisfy cellular carbon requirements when simple sugars such as glucose or fructose are not available


# TCA Enzymes in astrocytes
**Human**
CS ACO1 ACO2 IDH1 IDH2 IDH3A IDH3G IDH3B OGDH DLST DLD SUCLG1 SUCLG2 SUCLA2 SDHA SDHB SDHC SDHD SDHAF1 SDHAF2 SDHAF3 SDHAF4 FH MDH1 MDH2

**Mouse**
IDH3G = Idh3g & 4933405O20Rik
FH = Fh1

cs aco1 aco2 idh1 idh2 idh3a idh3g idh3b ogdh dlst dld suclg1 suclg2 sucla2 sdha sdhb sdhc sdhd sdhaf1 sdhaf2 sdhaf3 sdhaf4 fh1 mdh1 mdh2

**Citrate synthase**
*irreversible*
Oxaloacetate + Acetyl CoA + H2O :arrow_right: Citrate
CS

**Aconitase**
Citrate :arrow_right: cis-Aconitate + H2O
cis-Aconitate :arrow_right: Isocitrate
ACO1
ACO2
+many pseudo-genes

**Isocitrate dehydrogenase**
Isocitrate + NAD+ :arrow_right: Oxalosuccinate + NADH + H +
Oxalosuccinate :arrow_right: α-Ketoglutarate + CO2
*rate-limiting, irreversible stage*
IDH1 (very specific to Oligos)
IDH2
IDH3A
IDH3G
IDH3B

**Oxoglutarate dehydrogenase complex**
α-Ketoglutarate + NAD+ + CoA-SH :arrow_right: Succinyl-CoA + NADH + H+ + CO2
*irreversible stage*
OGDH
DLST
DLD

*BBOX CONVERTS Oxoglutarate to succinate*
*BBOX CONVERTS γ-butyrobetaine to Carnitine*

**Succinyl-CoA synthetase**
Succinyl-CoA + GDP + Pi :arrow_right: Succinate + CoA-SH + GTP
SUCLG1 Succinyl-CoA binding Alpha-unit
SUCLG2 (GTP/GDP forming) Beta-unit
SUCLA2 (ATP/ADP forming) Beta-unit

**Succinate dehydrogenase complex** (SDH) Complex II
Succinate :arrow_right: Fumarate
(Succinate + ubiquinone (Q) :arrow_right: Fumarate + ubiquinol (QH2))
SDHA
SDHB
SDHC
SDHD
SDHAF1 SDHAF2 SDHAF3 SDHAF4 = Assembly factors

[Also see slides Astrocyte TCA](https://docs.google.com/presentation/d/1OBWe0RZk7GlKqi6yrUXaou-iYRY9BSwG5OhjTSUT1u0/edit#slide=id.g12fd9a739a2_1_0)

**Fumarase**
Fumarate + H2O :arrow_right: L-Malate
FH

**Malate dehydrogenase**
L-Malate + NAD+ :arrow_right: Oxaloacetate + NADH + H+
MDH1
MDH2







**Red arrows = irreversible. Blue Astrocyte specific**

**They are not higher or lower in males/females** (campbell)
**They do not change with diet.** (campbell)

Most interesting are:

**Idh1,2** "irreversible conversion of isocitrate to alpha-ketoglutarate" Oligos do this differently?

**Suclg2** "Succinyl-CoA to succinate"
astrocytes does this different from most cells

= Astrocytic TCA activity - formation of succinate produces GTP.

Succinate in astrocytes cannot be converted into Fumarate. (due to lacking SDHD + SDHAF4 complex II)

=> QUESTION: What happens to Succinate in astrocytes?

# GTP formed in TCA

# NMEs // NDPKs

[See also Purines - Phosphor](https://hackmd.io/@sholmqvist/BJpKurTBB/https%3A%2F%2Fhackmd.io%2F4YTH0e5OSbixap5-w-F-wA#Purines)

[See also GTP - Phosphor](https://hackmd.io/@sholmqvist/BJpKurTBB/https%3A%2F%2Fhackmd.io%2F4YTH0e5OSbixap5-w-F-wA#GTP)

==suclg2 mainyl in astrocytes - not in neurons
This means neurons are dependent on de novo GTP synthesis (?) and NMEs for generating GTP. This has implications for G-protein signaling==

NME1 NME2 NME3 NME4 NME5 NME6 NME7 NME8 NME9

"Nucleoside-diphosphate kinases"

NME1 // NDPKA

**"Converts between ATP and GTP"**

GTP can be converted to ATP by NMEs
NME/NM23 Nucleoside Diphosphate Kinase

**Nucleoside diphosphate kinase (NDK) complex** exists as a hexamer composed of 'A' (encoded by this gene) and 'B' (encoded by NME2) isoforms

Major role in the synthesis of nucleoside triphosphates other than ATP

**NME1** // NDPKA
Possesses nucleoside-diphosphate kinase, serine/threonine-specific protein kinase, geranyl and farnesyl pyrophosphate kinase, histidine protein kinase and 3'-5' exonuclease activities.

**NME2** // NDPKB
Negatively regulates Rho activity by interacting with AKAP13/LBC (PubMed:15249197).Acts as a transcriptional activator of the MYC gene; binds DNA non-specifically (PubMed:8392752, PubMed:19435876). Binds to both single-stranded guanine- and cytosine-rich strands within the nuclease hypersensitive element (NHE) III(1) region of the MYC gene promoter.
Exhibits histidine protein kinase activity (PubMed:20946858)

**NME3** // NDPKC
Probably has a role in normal hematopoiesis by inhibition of granulocyte differentiation and induction of apoptosis.

**NME4** // NDPKD
Binds to anionic phospholipids, predominantly to cardiolipin; the binding inhibits its phosphotransfer activity (PubMed:18635542, PubMed:23150663)
Acts as mitochondria-specific NDK; its association with cardiolipin-containing mitochondrial inner membrane is coupled to respiration suggesting that ADP locally regenerated in the mitochondrion innermembrane space by its activity is directly taken up via ANT ADP/ATP translocase into the matrix space to stimulate respiratory ATP regeneration (PubMed:18635542).

**NME5** Tanycytes?
Does not seem to have NDK kinase activity.
Confers protection from cell death by Bax and **alters the cellular levels** of several antioxidant enzymes including **Gpx5**. May play a role in spermiogenesis by increasing the ability of late-stage spermatids to eliminate reactive oxygen species

[See Gpx5 in Glutathione](https://hackmd.io/@sholmqvist/BJpKurTBB/%2FsZITyKgiSTGGEHEpKig2cw#mammalian-Gpxs)

**NME6**
Inhibitor of p53-induced apoptosis.

**NME7**
localizes to the centrosome and functions as a component of the gamma-tubulin ring complex which plays a role in microtubule organization

**NME8**
Probably required during the final stages of sperm tail maturation in the testis and/or epididymis
May be involved in the reduction of disulfide bonds within the sperm FS components. In vitro, it has neither NDP kinase nor reducing activity on disulfide bonds.

Mutations in this gene are implicated in primary ciliary dyskinesia type 6



# A NAÏVE LOOK AT THE Standard model of CITRIC ACID CYCLE - Which way does it turn in hypoxia?
https://onlinelibrary.wiley.com/doi/full/10.1002/jnr.23196
# Is oxygen or glucose always available in equal amounts? What happens when enough oxygen is not around? When enough glucose is not around?

Only two reactions of TCA are not unidirectional CS and α-ketoglutarate dehydrogenase complex (OGDC = OGDH, DLST, DLD)

It is unfortunate that the depiction of the citric acid cycle as a unidirectional pathway is widespread, whereas in fact the irreversible reactions are only those catalyzed by **citrate synthase** and the **α‐ketoglutarate dehydrogenase** complex


[Astrocytes are high in CS but low in OGDH-Complex]
[Astrocytes are high in CS but low in OGDH-Complex]

- CS citrate synthase E.C. 2.3.3.1
- OGDC // Oxoglutarate dehydrogenase = complex α-ketoglutarate dehydrogenase complex
- OGDH
- DLST
- DLD
- Oxoglutarate dehydrogenase is a key control point in the citric acid cycle. It is inhibited by its products, succinyl CoA and NADH
- A high energy charge in the cell will also be inhibitive. ADP and calcium ions are allosteric activators of the enzyme
- By controlling the amount of available reducing equivalents generated by the Krebs cycle, Oxoglutarate dehydrogenase has a downstream regulatory effect on oxidative phosphorylation and ATP production.
- In biochemistry, the term reducing equivalent refers to any of a number of chemical species which transfer the equivalent of one electron in redox reactions.
- Oxoglutarate dehydrogenase is considered to be a redox sensor in the mitochondria, In the presence of a high concentration of free radical species, Oxoglutarate dehydrogenase undergoes fully reversible free radical mediated inhibition.
- [Bbox1 omits this and converts to succinate regardless or maybe not since Bbox1 only acts on extramitrochondrial alpha-ketoglutarate.]

Ironically, Krebs himself was proving his theory on the cycle by fighting opposing views of other researchers who ignored the reversibility of several steps. Below is an excerpt from a reply to the criticisms of F. L. Breusch and of J. Thomas (Breusch, 1937, 1939; Thomas, 1939):

Several of the criticisms arise solely from a misinterpretation of the theory. Thomas as well as Breusch draw incorrect conclusions from the theory and argue against the theory when these conclusions are not confirmed by their experiments.
This applies, for instance, to Thomas's statement that the formation of malate from oxaloacetate must be inhibited by malonate if the citric acid cycle is correct. In actual fact it was expressly stated that the reactions succinate ↔ fumarate ↔ malate ↔ oxaloacetate are reversible, and that oxaloacetate can be converted into the other C4 dicarboxylic acids in two ways, either anaerobically, or aerobically, the first being unaffected by malonate.
Thomas omitted the reversibility symbol (↔) in his version of the theory and hence reaches incorrect conclusions (Krebs, 1940b).

## The anaerobic TCA

* oxaloacetate can be anaerobically converted into malate, fumarate, or succinate in the presence of
**malonate**, a competitive inhibitor of succinate dehydrogenase 
chemical malonate decreases cellular respiration

These are organic compounds containing exactly two carboxylic acid groups. Malonic acid is a very hydrophobic molecule, practically insoluble (in water), and relatively neutral. Malonic acid exists in all living species, ranging from bacteria to humans. Within humans, malonic acid participates in a number of enzymatic reactions

#### Succinyl coenzyme A synthetase (SCS, also known as succinyl-CoA synthetase or succinate thiokinase or succinate-CoA ligase)

- The reaction is bidirectional

Succinyl CoA + Pi + NDP ↔ Succinate + CoA + NTP

**Sucla2**
Succinate--CoA Ligase [ADP-Forming] Subunit Beta, Mitochondrial
Hs neurons (Oldham)

**Suclg1** // **Sucla1**
Succinate--CoA Ligase [ADP/GDP-Forming] Subunit Alpha, Mitochondrial

**Suclg2**
Succinate--CoA Ligase [GDP-Forming] Subunit Beta, Mitochondrial

Hs astro (oldham)
suclg2-as1 only in astro (lucas)
Trend for tanycytes (campbell)

[Dobolyi et al 2015](10.1007/s10863-014-9586-4)

We have recently shown that the ATP-forming SUCLA2 subunit of succinyl-CoA ligase, an enzyme of the citric acid cycle, is exclusively expressed in neurons of the human cerebral cortex

Western blotting of post mortem samples revealed a minor SUCLG2 immunoreactivity

The absence of SUCLA2 and SUCLG2 in human glia is in compliance with the presence of alternative pathways occurring in these cells, namely the **GABA shunt** and **ketone body metabolism** which do not require succinyl CoA ligase activity, and **glutamate dehydrogenase 1**, an enzyme exhibiting exquisite sensitivity to inhibition by GTP.

**GLUD1 // glutamate dehydrogenase 1**
(GLUD1 key role in nitrogen and glutamate (Glu) metabolism and energy homeostasis)

GLUD1 is thought to be involved in insulin secretion mechanisms

This dehydrogenase is expressed at high levels in liver, brain, pancreas and kidney, but not in muscle

In nervous tissue, where glutamate is present in concentrations higher than in the other tissues, GLUD1 appears to function in both the synthesis and the catabolism of glutamate and perhaps in ammonia detoxification

The GTP-binding site is considered to be the "sensor" that turns the enzyme off when the cell is at a high energy state

# Citric Acid Cycle - KREBS
:::info

Carbohydrate, protein lipid pathways
:::

Most high school textbooks reflect this period of biochemistry knowledge and do not emphasize how the lipid and amino acid degradation pathways converge on the TCA cycle.

::: info
Questions
Transport of intermediates closer to the TCA?
E.g.
1. Acetyl-CoA (joint step of lipid and carbon metabolism)
2. [Citrate ](https://en.wikipedia.org/wiki/Acetyl-CoA#Extramitochondrial)Exported from Organelles. broken into 3. & 4.
3. Acetyl-CoA
4. Oxaloacetate
Better than transporting Lactate to the neurons?

Aminoacids can fuel the most energy producing steps (puruvate oxidation & citric acid cycle).

In low oxygenation anaerobic metabolism will generate lactate and pyruvate in surplus. lactate

In low glucose and/or high energy-needs metabolism.
If glucose cant be delivered fast enough and there is no fattyacids to spare/dismantle.
Then the evenmore abundant amino acids will be sucked out of the system.

:::

Aerobic

Link to Khan academy
https://www.khanacademy.org/science/biology/cellular-respiration-and-fermentation/variations-on-cellular-respiration/a/connections-between-cellular-respiration-and-other-pathways

:::info
TCA Inputs
:::
:::info
TCA Inputs - Carbohydrates
:::
:::info
TCA Inputs - Proteins - Glutamate
:::
:::info
TCA Inputs - Lipids
:::

Link to regulation of TCA ATP/AMP/
https://www.khanacademy.org/science/biology/cellular-respiration-and-fermentation/variations-on-cellular-respiration/a/regulation-of-cellular-respiration
TCA - regulation

"Oh Can I Keep Some Succinate For Myself?"

What is the effect of high carbohydrates and stress? over stimulation?

### Enzymes
Malatedehydrogenase (cytosol)

Enzyme that reversibly catalyzes the oxidation of malate to oxaloacetate

MDH1 - Hs neurons
MDH2 - all

Low in astrocytes

## Oxaloacetate
Part of TCA and Urea cycle

Can be produced from pyruvate by using ATP avoiding formation of acetyl-CoA. (doesnt seem to happen in neurons)

Can form PEP last step before pyruvate in Glycolysis / first step in gluconeogenesis.
oxaloacetate + GTP => phosphoenolpyruvate + GDP + CO2 [pck1]

oxaloacetate + glutamate <=> aspartate + alpha-ketoglutarate [GOT1]

## Phosphoenolpyruvate = PEP
*Enol of pyruvate and phosphate*
- Will only be around if very high levels of phosphate.
- Has the highest-energy phosphate bond found (deltaG −61.9 kJ/mol) in organisms
- Involved in glycolysis and gluconeogenesis
- - Results from the fact that the phosphorylated compound is "trapped" in the less thermodynamically favorable enol form.
- After dephosphorylation it can assume the keto form
- In glycolysis synthesised by Phosphopyruvate hydratase (Enolase) = penultimate step of glycolysis
PEP
- Synthesised by Enolase
ENO1 high in human astro.
ENO2 high in mouse neurons (increased in adult mice astro)
ENO3 low
ENO4 low (high in ependymal and choroid cells)

[#Slc1a3 takes up not only glutamate it also absorbs aspartate. Are neurons releasing aspartate?]

# enolases
The enolase superfamily includes enzymes that catalyse a wide variety of reactions and performing diverse roles in metabolism. However, the reactions catalysed share the common chemical step of abstraction of a proton from a carbon adjacent to a carboxylic acid and a requirement of a divalent metal ion[1]. This diversity of functions is in contrast to many families of enzymes whose members catalyse similar chemical reactions on different substrates.

# astrocytes neurons and TCA

## citrate
can be trasnported between cells (? ref)

* Citrate can be transported out of the mitochondria and into the cytoplasm, then broken down into acetyl-CoA for fatty acid synthesis, and into oxaloacetate

* Citrate controls the non-reversible step in fatty acid synthesis

Citrate is a positive modulator of this conversion, and allosterically regulates the enzyme acetyl-CoA carboxylase, which is the regulating enzyme in the conversion of acetyl-CoA into malonyl-CoA (the commitment step in fatty acid synthesis)

ACACA - enriched in human neurons.
ACACB - only expressed in some cells. enrhiched in human astrocytes.

* High concentrations of cytosolic citrate can inhibit phosphofructokinase, the catalyst of a rate-limiting step of glycolysis

CS - in neurons

Aconitase = ACO1 - low in neurons, higher in astro high in new oligos.

ACLY - in neurons
ADP + Phosphate + Acetyl-CoA + Oxalacetic acid → Adenosine triphosphate + Citric acid + Coenzyme A

Idh2 expressed by astro.

[#"astrocytes takes care of all the citrate produced from the TCA in neurons. This relives glycolysis from the negative feedback effect of citrate"].

Alpha-ketoglutarate.

Glutamate, GABA (+ Aspartate? and citrate) is taken up by astrocytes.

Glutamine is made in astro (GS)

Glutamine transferred to neurons.

There Glutamine converts to Glutamate
and glutamate to α-ketoglurate (2-oxoglutarate) by Glutamate dehydrogenase (GLDH/GDH/*GLUD1*).

2oxoglutarate transporter slc25a11 is not expressed by astrocytes.

Glutamte is then decarboxylated to GABA. GAD1 GAD2

## Oxaloacetate
can be transported between cells (? ref)

Part in gluconeogenesis, the urea cycle & the glyoxylate cycle (might not happen in vertebrates - debated
CLYBL, MS, ICL)

Oxaloacetate is also a potent inhibitor of complex II

Aspartate and alanine are formed from oxaloacetate and pyruvate, respectively, by transamination from glutamate (the n.t.)


# The reverse TCA


Reversible cytosolic alanine aminotransferase reaction

Performed by ALAT.
L-alanine + α-ketoglutarate ⇌ pyruvate + L-glutamate