Abstract inhibition of CAC enzymes. Step Reactants Enzyme Products

Abstract

The citric acid cycle (CAC), also known as the Krebs cycle
and the tricarboxylic acid cycle, is a stage of cellular respiration. The role
of the cycle is to oxidise fuel molecules and to provide precursors in the form
of carbon skeletons for other metabolic pathways (e.g. fatty acid synthesis,
amino acid synthesis etc.). The CAC is described as amphibolic, as it has both
catabolic and anabolic functions. Catabolism is the breaking down of substances
to provide chemically available energy (e.g. ATP) and/or to generate
intermediates used in anabolic reactions, whereas anabolism involves the
formation of complex molecules from simpler ones and the storage of energy. The
cycle can be regulated to either increase or decrease the rate of reaction, and
regulation is often carried out via allosteric inhibition of CAC enzymes.

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Step

Reactants

Enzyme

Products

Reaction
type

1

Acetyl CoA (2C), Oxaloacetate (4C) and H2O

Citrate synthase

Citrate (6C), CoA and H+

Aldol condensation and hydrolysis

 
2
 

Citrate (6C)

Aconitase

Isocitrate (6C)

Dehydrating and then rehydrating

 
3

Isocitrate (6C) and NAD+

Isocitrate dehydrogenase

?-Ketoglutarate (5C), CO2 and NADH

Oxidative decarboxylation

 
 
4

?-Ketoglutarate (5C), NAD+ and CoA

?-Ketoglutarate dehydrogenase complex

Succinyl CoA (4C), CO2 and NADH

Oxidative decarboxylation

 
5
 

Succinyl CoA (4C), Pi, GDP and H2O

Succinyl CoA synthase

Succinate (4C), GTP and CoA

Substrate-level phosphorylation

 
6

Succinate (4C) and FAD

Succinnate dehydrogenase

Fumerate (4C) and FADH2

Oxidation

7

Fumerate (4C) and H2O

Fumerase

L- Malate (4C)

Hydration

 
8

L-Malate (4C) and NAD+

Malate dehydrogenase

Oxaloacetate (4C), NAD and H+

Oxidation

Introduction

The citric
acid cycle was discovered in 1937 by Hans Krebs and William Arthur Johnson
(Krebs and Johnson, 1937). The cycle has been described as “the hub of the
metabolic wheel” due to its role of oxidising fuel molecules, and providing
precursors involved in other metabolic pathways. The CAC is a stage in the
process of respiration that occurs in the mitochondrial matrix, and involves a
series of oxidation-reduction reactions. It is preceded by glycolysis and the
link reaction. See Table 1 for a summary of the eight principle steps of the
cycle. This essay will discuss how the CAC links to oxidative phosphorylation
and other such metabolic pathways, the cycle’s role as a source of precursors,
how intermediates in the cycle are replenished, how the cycle is regulated and
finally how disease, nutrition and harmful substances can affect the cycle.

 

Links to other metabolic pathways

The citric acid cycle is linked to many metabolic pathways,
as a part of its role as “the hub of the metabolic wheel”. As a stage in the
process of respiration the cycle is linked to oxidative phosphorylation which
follows it. 

The NADH and FADH2 produced by CAC links the cycle
to oxidative phosphorylation. During oxidative phosphorylation, electrons
derived from NADH and FADH2 are oxidised and the energy released
from these reactions is used to drive the synthesis of ATP from ADP. The
electrons travel through a number of carriers which make up the electron
transport chain (Cooper, 2000). See Figure 1 for a summary of electron movement
in oxidative phosphorylation.  

The cycle is linked
to the biosynthetic pathways of amino acids, nucleotide bases and porphyrin
production because intermediates of CAC are precursors involved in these
metabolic pathways (see figure 2). The cycle as a source of precursors shows
the anabolic properties of the cycle. Anabolic reactions involve the formation
of complex molecules from simpler ones and the storage of energy. Anabolism is
powered by catabolism (Lawrence, 2000; Nicholls and Ferguson, 2009).

The intermediate ?-Ketoglutarate is the carbon skeleton of glutamate,
glutamine, proline and arginine. ?-Ketoglutarate acquires its amino groups from
the transamination of glutamate, which converts glutamate into ?-Ketoglutarate.
As an intermediate in the CAC ?-Ketoglutarate is converted into glutamate by
reductive amination. Glutamate is a precursor of glutamine, proline and
arginine. Aspartate is synthesised from oxaloacetate by transamination from
glutamate. Asparagine, methionine, threonine and lysine are synthesised from
aspartate (see figure 3). (Stryer et al., 2015; Lehninger et al., 1993).  

The citric acid cycle also has a
role in producing the carbon skeletons of purines and pyrimidines. The amino
acids aspartate which is synthesised from the intermediate oxaloacetate and
glutamate which is synthesised from the intermediate ?-Ketoglutarate are components
of the purine nucleotide cycle, see figure 4, which also produces the CAC
intermediate fumarate. As seen in figure 5, glutamate and aspartate is also
involved in pyrimidine synthesis (Moffatt and Ashihara, 2002).

 

 

 

 

 

 

 

                         

 

 

 

Porphyrins are a group of pigments that have a heterocyclic
structure formed from four pyrrole rings linked by four methylene groups, such
as heme and chlorophyll (Collinsdictionary.com, 2017). The CAC is linked to
porphyrin synthesis as the succinyl CoA (a CAC intermediate) is the carbon
skeleton of porphyrin. Glycine reacts with succinyl CoA to begin the porphyrin
biosynthetic pathway (Lehninger et al., 1993).

Finally, the CAC is linked to the fatty acid biosynthetic
pathway in a process called the citrate shuttle. The acetyl CoA produced in the
link reaction has to be transported from the mitochondrion to the cytoplasm in
order for fatty acid synthesis to take place, and this is done by the citrate
shuttle. In the CAC, oxaloacetate reacts with acetyl CoA to produce citrate,
which is then transported into the cytosol by the enzyme tricarboxylate
translocase. In the cytosol, citrate is cleaved back to oxaloacetate and acetyl
CoA, which can then be used in fatty acid biosynthesis (Medh, n.d.).

Reactions that
replenish cycle intermediates 

The process of anaplerosis is the replacement of CAC
intermediates that are removed from the cycle, and this process allows the
continuation of the cycle (Owen et al., 2002). In Figure 2 the red arrows show the
anaplerotic reactions that replenish depleted intermediates.

Oxaloacetate is replenished via the carboxylation of
pyruvate, which is catalysed by the enzyme pyruvate carboxylase. The
carboxylation of pyruvate also requires biotin as it is a prosthetic group of
pyruvate carboxylase, hence biotin is a component of the enzyme’s quaternary
structure (Stryer et al., 2015; Lehninger et al., 1993).

See table 2 for a summary of
anaplerotic reactions that replenish cycle intermediates.

The products of the metabolic pathways discussed in the
previous section can replenish cycle intermediates. For example, amino acids
produced from CAC intermediates can also be used to regenerate such
intermediates (see table 3). 

Amino acids converted to pyruvate ???

Alanine, serine, glycine, threonine, cysteine, tryptophan

Amino acids converted to oxaloacetate ???

Aspartate, asparagine

Amino acids converted to ?-ketoglutarate

Glutamate, glutamine, proline, histidine, arginine

Amino acids converted to fumarate ???

Phenylalanine, tyrosine

Amino acids converted to succinyl-CoA ???

Methionine, isoleucine, valine

Amino acids converted to acetyl-CoA ???

Leucine, isoleucine, lysine, phenylalanine, tyrosine, tryptophan,
threonine

As well as this, the purine nucleotide cycle replenishes the
intermediate fumarate, as can be seen in figure 4.  The carbon skeleton of aspartate leaves the
purine nucleotide cycle as fumarate in the cytosol, which is then transported
to the mitochondria and is converted into oxaloacetate in CAC. (Arinze, 2005).

Regulation of the
cycle

There are a number of mechanisms that play a role in
regulating the CAC, controlling the rate at which it oxidises fuel molecules.
One such mechanism is inhibiting the enzyme that catalyses the production of
acetyl CoA which will result in less citrate being produced. In vertebrates the
pyruvate dehydrogenase complex reaction, in which pyruvate is converted to
acetyl CoA, is allosterically inhibited when there are high ratios of ATP to
ADP, NADH to NAD+ and acetyl-CoA to CoA. Covalent protein modification can also
be used to regulate the pyruvate dehydrogenase complex. The complex is
inhibited by the phosphorylation of the component E1 by a kinase that
switches off the activity of the complex. High concentrations of reaction
products inhibit the reaction. Acetyl CoA inhibits the transacetylase component
E2 and NADH inhibits the dihydrolipoyl dehydrogenase E3.

Enzymes involved in CAC can be inhibited in order to
regulate the cycle. Citrate synthase catalyses the reaction between
oxaloacetate and acetyl CoA. The enzyme is inhibited by high concentrations of
ATP because ATP is an allosteric inhibitor of citrate synthase. The enzyme
isocitrate dehydrogenase catalyses the conversion of citrate to ?-ketoglutarate.
The enzyme is inhibited by a high concentration of NADH, which is a product of
the conversion, because NADH displaces NAD+. However, the enzyme is
allosterically stimulated by ADP because ADH increases isocitrate dehydrogenase’s
affinity for substrates. Succinyl CoA and NADH inhibit the enzyme ?-ketoglutarate
dehydrogenase as they are products of the reaction that the enzyme catalyses.

Finally, calcium is another important regulator of the CAC.
Calcium ions activate pyruvate dehydrogenase, stimulating the production of
acetyl CoA. Calcium ions also activate isocitrate dehydrogenase and
?-ketoglutarate dehydrogenase, increasing the rate of the reactions those
enzymes catalyse (Stryer et al., 2015; Lehninger et al., 1993; Farrar Stoakes,
2017)

The effect of
disease, nutrition and harmful substances on the citric acid cycle

Genetic diseases can negatively affect CAC function,
preventing it from carrying out its role as “the hub of the metabolic wheel”. Although
rare, there are cases of patients with defects of CAC enzymes which are due to deletion
mutations. Patients have been observed to have defective ?-ketoglutarate
dehydrogenase, succinate dehydrogenase and fumarase enzymes, often
alongside defective enzymes in other metabolic pathways. Fumarase and ?-ketoglutarate
dehydrogenase defects are usually detected within 12 months of birth, whereas succinate
dehydrogenase deficiencies are often diagnosed after several years of life, and
even in some cases in adulthood (Rustin et al., 1997).

Nutritional deficiencies can also interfere with cycle
function. CAC will not occur if an individual is deficient in thiamin,
riboflavin, niacin, lipoic acid, and pantothenic acid because these are
co-factors required in the conversion of pyruvate to acetyl CoA. If acetyl CoA
is not produced, then CAC will be inhibited as oxaloacetate will accumulate (Dean
and English, 2013).

There are also some poisons that inhibit the CAC. Fluoroacetate
(a pesticide) reacts with oxaloacetate to form fluorocitrate which inhibits
aconitase, causing citrate to accumulate as the aconitase enzyme catalyses the
conversion of citrate to isocitrate. Arsenic inhibits ? -ketoglutarate
dehydrogenase, causing a build up of ?-ketoglutarate as it conversion to
succinyl CoA is prevented as the reaction cannot be catalysed by ?
-ketoglutarate dehydrogenase (Akram, 2013).

Conclusion

In conclusion, describing the citric acid cycle as “the hub
of the metabolic wheel” is an accurate statement due to its involvement in a
wide range of metabolic pathways. The cycle is linked to many pathways
including oxidative phosphorylation and biosynthetic pathways that the CAC
provides precursors for and receives intermediates from. The cycle is regulated
by its own products synthesised via the oxidation of fuel molecules (e.g.
NADH), by enzymes involved in the cycle and by other molecules such as calcium.
This regulation has a knock-on effect on its role of oxidising fuel molecules
and providing carbon skeletons as regulation can increase or decrease the rate
at which the cycle takes place. The role of the cycle can be impaired by
genetic diseases that affect CAC enzymes, by nutritional deficiencies and by
certain poisons. 

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