Biochemistry- the citric acid cycle

Question 1

Give an overview of the citric acid cycle

Answer 1

The primary purpose of the citric acid cycle (also called the tricarboxylic
acid cycle or Krebs cycle) is to oxidise acetyl CoA, producing reduced
coenzymes, that are then oxidised in the electron transport chain to
produce energy.
 The citric acid cycle provides precursors for synthesis of a variety of other
compounds.
 The most notable feature of the citric acid cycle is that it is closely linked
to numerous metabolic pathways, a metabolic hub.
 The activity of the citric acid cycle is very sensitive cellular conditions,
increasing or decreasing its rate almost instantaneously in response to
the cell’s needs.

Question 2

What are the sources of Acetyl CoA

Answer 2

Fatty acids are broken down into acetyl CoA via β-oxidation. This primarily occurs in the fasting state, or during exercise.
 Some types of amino acids can be converted into acetyl CoA, and used to fuel the citric acid cycle. These amino acids are
called ketogenic (as opposed to glucogenic) because they can be converted into ketone bodies (but not glucose).
 Sugars undergo glycolysis, producing pyruvate. Pyruvate can undergo oxidative decarboxylation into acetyl CoA. This
reaction is irreversible and the gluconeogenic potential of pyruvate is lost. This is most common during the fed state.

Question 3

What are the destinations of pyruvate

Answer 3

If glucose and oxygen are plentiful, and there is no need to get rid of nitrogen waste, pyruvate is broken down to acetyl CoA.
 Lactate is produced in the absence of oxygen, to regenerate NAD. The lactate is released into the blood, and the liver is used
to regenerate pyruvate. The reaction producing lactate allows glycolysis to occur at a very high rate.
 Pyruvate can be converted to oxaloacetate. Increasing oxaloacetate increases the rate of the citric acid cycle. Oxaloacetate
can be used to make glucose.
 Pyruvate can be converted to alanine, by adding nitrogen to it. Any tissue that needs to remove nitrogen waste does it
through producing alanine. When the liver removes nitrogen waste, it can take the alanine and convert to glucose and
excrete nitrogen waste.

Question 4

Describe thiamine (B1 deficiency)- Beri Beri

Answer 4

Thiamine is needed to make a cofactor of pyruvate dehydrogenase.
 Metabolism of pyruvate is very important for nerve cells, so the nervous system is strongly affected (e.g. tingling,
confusion).
 Dietary changes (e.g. switching from brown to white rice) can lead to deficiency, and some foods contain thiaminases.
 Infantile beri-beri seen in breast-fed babies of affected mothers at 2-3 months. Death may occur within minutes of sudden
onset of shock or respiratory distress.
 Thiamine injection can lead to recovery within 6 hours.

Question 5

What happens if you have a pyruvate dehydrogenase deficiency

Answer 5

Typically diagnosed in infancy leading to delayed development, reduced muscle tone, often with seizures.
 All patients with PDF deficiency have some form of neurological impairment. This is because the brain normally obtains all
of its energy from the aerobic oxidation of glucose.
 Foetuses have poor weight gain towards the last trimester of pregnancy, due to a lack of fatty acid synthesis.
 Because pyruvate cannot enter the citric acid cycle, it is converted to lactate. There are elevated levels of pyruvate and
lactate in the plasma, as pyruvate is not properly metabolised.
 Treatment: a ketogenic diet with <15% protein, <5% carbohydrate. This ensures that cells use acetyl CoA from fat metabolism.

Question 6

Summarise the citric acid cycle

Answer 6

Acetyl CoA enters the citric acid cycle by addition to oxaloacetate.
 2 carbons are lost as CO2 and oxaloacetate is reformed.
 During this process, NADH, FADH2 and GTP (or ATP) are produced.

Question 7

What are the key enzymes involved in the citric acid cycle

Answer 7

Citrate synthase
 Regulated enzyme
 Affected by: ADP:ATP ratio, NADH levels, product accumulation, substrate
availability (if [oxaloacetate] is low, then acetyl CoA cannot be metabolised in
the citric acid cycle).
 In the liver, this could result in directing acetyl CoA towards ketogenesis as acetyl CoA does not enter the citric acid cycle.

1.1.1. Fluoroacetates
 Fluoroacetates are toxic due to the lethal synthesis of
fluorocitrate, a suicide inhibitor of aconitase.
 Citrate synthase treats fluoroacetyl CoA the same as acetyl
CoA. It can then react with the next enzyme (aconitase) in
the citric acid cycle, permanently inhibiting it. Citrate levels
then increase, causing further inhibition of glycolysis and
fatty acid oxidation.
 Treatment: ethanol is metabolised to acetate, and competes with fluorocitrate to slow down the rate of inhibition.

1.2. Isocitrate dehydrogenase
 Regulated enzyme: extremely exothermic.
 This is an oxidative decarboxylation.
 Affected by: ADP:ATP and NAD:NADH ratios.
 In muscle, its activity is increased by Ca
2+. This allows more energy production during muscle contraction. In skeletal muscle
the [Ca
2+] increases during contraction which goes into the mitochondria and activates these enzymes.

1.3. 𝛂-ketoglutarate dehydrogenase
 Regulated enzyme
 This is an oxidative decarboxylation and uses the same mechanism as PDH
(coenzyme A is also a substrate).
 Affected by: AD:NADH ratio, product inhibition, Ca
2+ in muscles.
 Because α-ketoglutarate is so similar to PDH, conditions that affects PDH often affect α-KGDH (e.g. beri-beri).

1.4. Succinate dehydrogenase
 Also complex II of the electron transport chain. Succinate is oxidised to
fumarate.
 FAD is covalently attached to the enzyme. It accepts electrons from succinate, and these electrons are passed to coenzyme
Q in the ETC.
 Other FAD containing enzymes (e.g. glycerol-3-P dehydrogenase and fatty acyl CoA dehydrogenase) also pass their electrons
directly to coenzyme Q.
 This enzyme is embedded within the inner mitochondrial membrane, while the other citric acid cycle are in the matrix.

Question 8

In summary, what are the products of the citric acid cycle

Answer 8

In combination with the electron transport chain (which requires
O2), the oxidative catabolism of acetyl CoA can produce 10 ATP
equivalents:
o 7.5 ATP equivalents from NADH
o 1.5 ATP equivalents from FADH2
o 1 ATP equivalent from GTP

Question 9

What are the intermediates in the citric acid cycle

Answer 9

In order for acetyl CoA to be metabolised at a fast rate, there must be enough oxaloacetate to react with (or any other
intermediates can also be converted to oxaloacetate).
 Anaplerotic reactions increase the number of citric acid cycle intermediates, adding to the amount of carbon in the cycle.
Addition of acetyl CoA does not count, because it results in two carbons being lost anyway (not the same ones).
 Intermediates are also drawn off for synthesis of other compounds, if not replaced, this will decrease the rate at which acetyl CoA can be oxidised.

Question 10

What are anaplerotic reactions

Answer 10

There are several reaction in which compounds are converted into intermediates of
the citric acid cycle.
 Pyruvate carboxylase: this is useful when pyruvate is building up because
acetyl CoA cannot be metabolised quickly enough, so some pyruvate is
used to increase the activity of the citric acid cycle by increasing the
number of intermediates.
 Aspartate aminotransferase and glutamate dehydrogenase: this is useful
when using amino acids as a fuel. Also, by converting amino acid skeletons
into citric acid cycle intermediates, they can be used to make glucose.

Question 11

Describe the regulation of the citric acid cycle

Answer 11

The citric acid cycle occurs all in the mitochondria.
 If the cell is using more energy, then it is more active (e.g. during exercise (muscle cells)).
 To increase the activity:
o Increase the activity of the enzymes.
o Increase the supply of acetyl CoA.
o Increase the concentration of intermediates.
 Fed state: glycolysis is increased, so acetyl CoA comes from sugars.
 Fasting state: in most tissues, acetyl CoA is supplied from fatty acids, as glycolysis is inhibited.
 Exercise: in muscles, citric acid cycle enzymes are stimulated, acetyl CoA supply is increased (from fatty acids and glucose)
and the concentration of intermediates is also increased.