Citric Acid Cycle Shunts and Bypasses
Evolution has allowed variants in the citric acid cycle to produce new functionalities in organisms. Let's consider a few.
What if you were a microorganism that has evolved to use acetate (2C) as a source (if not the sole source) of energy? Remember that the citric acid cycle is also used to generate metabolites for anaplerotic reactions (from the Greek ἀνά= 'up' and πληρόω= 'to fill'). In the full citric acid cycle, activated acetate (2C) reacts with oxaloacetate (4) to produce the 6C molecule citric acid. As the cycle continues back to oxaloacetate, 2 Cs are lost as CO2, so in sum for 1 turn of the cycle:
oxaloacetate (4C) + acetyl-CoA (2C) → Citrate (6C) → → → succinate (4C) + 2 CO2 (2C).
In the process, oxidation reactions occur leading to the formation of NADH and FADH2 and metabolites formed can be withdrawn for biosynthesis (OAA, αKG and succinyl CoA).
An alternative "cycle" would be to eliminate the two decarboxylation reactions and generate a 4C molecule (succinate) and one 2C metabolite (glyoxylate), which could react with another activated acetate (acetyl-CoA) to form oxalacetate (or more precisely malate which can be oxidized to it). Hence two acetates are required, but if you are an organisms adapted to use this as a metabolic energy source, it's no problem. Here is the next reaction
oxaloacetate (4C) + acetyl-CoA (2C) → Citrate (6C) → → succinate (4C) + glyoxylate (2C);
glyoxylate (2C) + acetyl-CoA (2C) → malate (4C )
This reactions would act as a metabolic shunt, altering the flow of metabolites by bypassing part of the citric acid cycle. The shunt, which is found in many microorganisms and some plants is called the glyoxylate shunt, which is shown in the figure below.
Why is this so cool? First consider this observation about the citric acid cycle. One acetate, in the form of acetyl-CoA, is need to form one oxalacetate in one turn of the cycle, which in turn is needed in the next turn of the cycle. Hence acetyl-CoA cannot, in net fashion, be used to synthesize oxalacetate. We saw previously that oxalate is an intermediate in the synthesis of phosphoenolpyruvate from pyuvate in gluconeogenesis. Hence acetate can not be used to form glucose in net fashion. (Note: pyruvate dehydrogenase is not reversible). Another way to think about this is 2Cs enter the cycle as acetyl-CoA and 2 leave as CO2s so no net synthesis can occur. And, you are in trouble if you draw off oxaloacetate as well as α-ketoglutarate and succinate form anapleurotic reactions for biosynthesis as the needed metabolites for a cycle which consumes and produces on of each of these will be depleted if withdrawn
Now if you are an organism (many bacteria and plants) that has the glyoxylate shunt, you have no worries. A minicycle still occurs with the conversion of isocitrate to glyoxylate to malate and back around to oxaloacetate and citrate (for energy production) with the net production of 1 succinate (for biosynthesis). The only Cs "lost" from the mini cycle are in the form of succinate so they are not really lost as they are as 2 CO2s in the citric acid cycle since as they are used for biosynthesis.
The next reaction for the glyoxylate cycle is:
2 Acetyl-CoA + 2 NAD+ + 2 H2O → 2 succinate + 2 NADH + 2 CoASH + 2 H+ .
In plants, this reaction occurs in organelles called glyoxysomes. The succcinate can be converted through its continuation through the citric acid to oxaloacetate which can then form glucose and other carbohydrates through gluconeogenesis. Hence organisms that have the glyoxylate shunt (or cycle) can synthesize carbohydrate in net fashion from acetate, which can derive from fattty acid degradation as we will see in a subsequent chapter.
Now to operate the glyoxylate cycle efficiently, you do need to activate the carboxyl end of acetate. Plants and other organisms that can grow on acetate can produce acetyl-CoA needed for the above pathways from the following reaction catalyzed by acetyl-CoA synthase (i.e. not pyruvate dehydrogenase)
Acetate + CoASH + ATP → Acetyl-CoA + AMP + PPi
Note that one phosphoanhydride bond is cleave and one thioester bond is formed. These are both high energy with respect to their hydrolysis products so this reaction alone is not thermodynamically favored in any significant way. The hydrolysis of pyrophosphate (PPi) drives this reaction forward. An analogous reaction drives the creation of peptide bonds in protein as that reaction also uses ATP to create an activate mixed anhydride from the free carboxyl end
Now let's move to the brain where there is lots of chemistry happening as you read this page. Relatively high concentration of key neurotransmitters are required for neural function and these need to be maintained against metabolic losses. Glutamate is the principle excitatory neurotransmitter and GABA (δ-aminobutryic acid) is a principle inhibitory neurotransmitter. Both are maintained at high concentrations and are principle players in the GABA shunt. The figure below shows a part of the large pathway shown above, but in a more expanded form.
GABA is formed in the cytoplasm by decarboxylation of Glu by the enzyme glutamate decarboxylase, which appears to be expressed predominantly in neural tissue. It is metabolized by GABA transaminase but only if the compound from which the shunt starts, α-ketoglutarate, is present abundantly. This conserves the supply of GABA in the neuron. Stated in another way,the breakdown product of GABA, succinic semialdehyde, is formed only if GABA's precursor is present. Inhibitors of GABA aminotransferase are used to treat epilepsy. Another function of the shunt is that it effectively allows glutamate to loop into the cycle.
α-ketoglutarate ↔ Succinate Bypass
In anticipation of the section below, one could ask the following question: What happens when a step in the cycle is impaired or missing? Cyanobacteria, a key player in atmospheric O2 production and draw down of natural and anthroprogenic atmospheric CO2, were thought to have an incomplete citric cycle as they lack α-ketoglutarate dehydrogenase. They work around this issue by converting α-ketoglutarate to succinate directly using two enzymes, alpha-ketoglutarate decarboxylase and succinic semialdehyde dehydrogenase, as shown below. The first enzyme catalyses a non-oxidative decarboxylation of the substrate to succinic semialdehyde. This bypass is shown in the figure below. In addition, cyanobacteria also use the GABA shunt as a bypass for α-ketoglutarate dehydrogenase.
Here is yet another example. Each member of the cycle is an important member. Let's consider the first one, citrate. It is formed in the key step coupling the output of anaerobic metabolism of glucose, pyruvate) with the formation of citrate in the citric acid cycle. What happens if aerobic metabolism is impaired, as it would in hypoxic and anoxic conditions, or if mitochondria function is compromised or in disease states such as cancer? Might there be another way to form citrate? Turns out that there is. and it centers on α-ketoglutarate again. It is illustrated in the above figure.
Lets consider cancer which is characterized by a state of rapid proliferation of cells. This requires both energy and metabolic precursors for biosynthesis of carbohydrates, lipids and proteins. The citric acid cycle offers both. To accommodate this demand for efflux of citric acid cycle intermediates for reductive biosynthesis, large fluxes of glutamate (derived from abundant gluatamine) into the cycle are used in the form of α-ketoglutarate. In another anapleurotic reaction, citrate can be cleaved by citrate lyase to form acetyl-CoA which can be used for fatty acid synthesis needed by rapidly proliferating cells. To replenish the citrate, cancer cells convert α-ketoglutarate by reductive carboxylation to isocitrate by isocitrate dehydrogenase 2, which uses NADPH for the reduction reaction. That NADPH comes from the enzyme nicotinamide nucleotide transhydrogenase, which can intercovert matrix pools of NADH and NADPH using the collapse of a protein gradient across the mitochondria inner membrane (which we will study in a subsequent chapter).
H+(in) + NAD+ + NADPH ↔ H+(out) + NADH + NADP+
Note that both oxidative (α-ketoglutarate to succinyl CoA) and reductive reactions (α-ketoglutarate to isocitrate) occur in this process.
Variants of the TCA cycle in Microorganisms
It's very easy to be anthropocentric in constructing a biochemistry text as many who take the course are interest in human medicine. Since a human is an ecosystem of organisms with an expansive microbiome on their skin and in their gut, even from a human health perspective, it's is important to compare the same pathway in different organisms. It's also important to understand our role as a small part of a vast biosphere where our survival depends on other organisms, large and small.
In that light, let's consider the citric acid cycle of other organisms. It seems that most organism have the anerobic and universal glycolytic pathway. How about the aerobic citric acid cycle? These days of single cell genomic analysis makes it simple in principle to analyze the citric acid cycle genes of any organism. Variants of it are found in generally all aerobic organism and even some anaerobic one. Some subtle differences exist between eukaryotic and prokaryotic organisms. NAD+ is used as a substrate in the mammalian form of isocitrate dehydrogenase while prokaryotes use NADP+. An NAD+-dependent malate dehydrogenase is used in mammals while some prokaroytes use a different enzyme, a NADP+-dependent malate-quinone oxidoreductase. Lastly different enyzmes (and unfortunately with varying names) are used to convert succinyl-CoA to succinate. Plants and fungi use ADP as a substrate, mammals have two different enzymes often named different and for the reverse reaction as succinate-CoA ligase (ADP forming) and succinate-CoA ligase (GDP forming).
Analyzes shown that few bacteria have complete cycles. Of those with incomplete cycles, the early steps are least conserved, while the latter are most conserved. The cycle is used not only for oxidative production of energy but also generation of metabolites (α-ketoglutarate, oxalacetate and succinyl-CoA), which are pulled from the cycle for biosynthesis. Autotrophs that don't have a complete cycle can make those products from pyruvate. Consider α-ketoglutarate. Some make it through the clockwise oxidative reaction from citrate to α-ketoglutarate while some methanogenic archaea make it through counterclockwise reduction reactions. Comparative genomic analyses suggest, in accordance with this description, that the citric acid cycle probably arose from a "linear" oxidative pathways leading to α-ketoglutarate and a reductive one leading to succinyl CoA. Knowing the pathways in individual microorganisms can assist in the rational drug design of new antibiotics. The figure below, adapted from Huynen et al, shows a geometric analysis diagram of the citric acid cycle and variants in other organisms.
The α-ketoacid pathway - A primordial, prebiotic anabolic "TCA-like" pathway
You might be wondering after studying the complexity of pyruvate dehydrogenase and the citric acid cycle how the cycle might have originated. As discussed above, clues come from comparative genome analysis of genes encoding the enzymes for the reactions in a multitude of organisms. By looking at evolutionary changes in genes and functions for these reactions, one can obtain some ideas of the first enyzmes that evolved in the pathway. But what about an abiotic pathway that might have arisen before the first biological one? Stubbs et al have shown that the very simple molecules glyoxylate and pyruvate can react in mild aqueous conditions (pH 7 in 0.5 M phosphate buffer heated to 50 °C) to form,in a single pot, α-ketoacids similar to present citric acid cycle intermediates. From a kinetic perspective, they were formed in the reverse, counterclockwise reductive direction compared to the citric acid cycle (clockwise, oxidative). No metals or "enzymes" were needed for the transformations. Glyoxylate acts as a reducing agent and simultaneously the source of carbon atoms. Once these α-ketoacids form, they could be theoretically converted by transamination reactions to amino acids as they should be characterized by a Keq ≈1 and a ΔG0 ≈ 0.
The citric acid cycle is filled are carboxylic acids. Only the α-ketoacids (α-ketoglutarate and oxaloacetate) can readily form carbanions at the alpha carbons as their Hs are slightly acidic due to resonance stabiiization of the carbanion through and enolate tautomer. The others are electron rich so they are less acidic at the alpha carbons, discouraging deprotonation in the absence of harsh catalysts. So perhaps alternative functional groups were present in abiotic precursors. Perhaps they contained α-ketoacids similar to α-ketoglutarate, oxaloacetate and of course pyruvate. In addition, α-ketoacids have an electrophile in the form of the carbonyl carbon form C-C bond formation.
The figure below shows the reductive, counter-clockwise pathway they determined. Note that in both the glyoxylate "cycle" which has also been proposed as a abiotic pathway, and the α-ketoacid reductive pathway, no CO2 is lost in any step. That suggest that actual decarboxylations steps, although often favored thermodynamically, required the development of catalyst. The two α-ketoacids substrates in this pathway, glycoxylate (2Cs) and pyruvate (3Cs) are the smallest and most likely formed acids available.
The animated figure below shows the entire citric acid cycle (catabolic, oxidative, running clockwise), the glyoxylate shunt which bypasses many steps in the citric acid cycle, and the proposed prebiotic anabolic alpha-ketoacid pathway (bold green arrows) which essentially runs counterclockwise and is reductive.