Content:
1. Introduction to the Krebs cycle
2. Reactions of the Krebs cycle
3. Regulation of the Krebs cycle
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Introduction to the Krebs cycle
The Krebs cycle (KC, tricarboxylic acid cycle = TCA cycle) is a metabolic pathway localized in the mitochondrial matrix. One should easily deduce that every cell which possesses mitochondria has in physiologic conditions active the TCA cycle. There is one cell population however that lacks mitochondria – the erythrocytes. In the erythrocytes the TCA cycle does not take place. There is one important fact you should notice. The TCA cycle needs aerobic conditions for smooth course (the reason is below – Regulation of the Krebs cycle). The TCA cycle in cells that lack oxygen has limited velocity.
The TCA cycle performs many functions. In simple words the TCA cycle is heart of the energetic metabolism of the cell, i.e. almost all pathways of the energetic metabolism are connected to the TCA cycle. For example the ETC (electron transport chain), gluconeogenesis, transamination, deamination of amino acids or lipogenesis. It is quite obvious that there are both catabolic, and anabolic pathways thus it is not easy to state which one dominates (it is not possible to denote the TCA cycle as either catabolic, or anabolic). This is the reason why it is described as amphibolic pathway (for more details: Subchapter 2/2). Here is the partial list of some functions of the TCA cycle:
1) Oxidation of acetyl residues (supplied as acetyl-CoA (AcCoA))
In the TCA cycle takes place oxidation of acetyl residues (CH3-CO-) to CO2.
This process is source of reducing equivalents these are transferred on cofactors, NAD+ or FAD. This yields reduced forms – NADH and FADH2. NADH and FADH2 are so called reduced coenzymes. Reduced coenzymes enter ETC and their regeneration takes place. This regeneration is loss of reducing equivalents (i.e. electrons). Hence it is called reoxidation.
The reoxidation (regeneration) of reduced coenzymes is the process which connects the TCA cycle with the ETC. The TCA cycle is the main supplier of the reduced coenzymes for the ETC, thus the TCA cycle is very important source of the ATP (regardless fact that one turnover of the TCA cycle produces only one GTP)
2) Many catabolic pathways flow into the TCA cycle
Many catabolic pathways are source of the (1) intermediates of the TCA cycle, (2) pyr, (3) AcCoA. Their fate could be: (1) oxidation to CO2, (2) synthesis of other substances.
3)The TCA cycle provides precursors for many important anabolic pathways
Examples are (1) gluconeogenesis, (2) biosynthesis of tetrapyrroles (hem), (3) synthesis of amino acids (e.g. glutamate – it is the most abundant excitatory neurotransmitter in the brain), (4) source of AcCoA for fatty-acids synthesis.
4) The TCA cycle takes part in excretion of nitrogen
The TCA cycle is connected with urea cycle and glutamate synthesis. Both urea, and glutamate are two main forms used for excretion of amino acids derived nitrogen.
Historic correlation:
Krebs cycle is named after Sir Hans Adolf Krebs (1900-1981). Krebs German, later English, doctor and biochemist. In 1953 he won the Nobel Prize in Physiology and Medicine “for his discovery of the citric acid cycle”. The Nobel Prize was given to him and German, later American, biochemist Fritz Albert Lipmann, who got it “for his discovery of co-enzyme A and its importance for intermediary metabolism”.
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Reactions of the TCA cycle
The overall equation for the TCA cycle is as follows:
CH3-CO~SCoA + 3NAD+ + FAD + GDP + Pi + 2H2O → 2CO2 + 3NADH + FADH2 + GTP
AcCoA provides acetyl residues for the TCA cycle. Majority of it is from (1) the β-oxidation of fatty acids and (2) the pyruvate dehydrogenase reaction. Both these pathways take place in mitochondrial matrix.
Pyruvate dehydrogenase reaction
This reaction is irreversible oxidative decarboxylation of pyruvate. Equation follows:
CH3-CO-COOH + NAD+ + HSCoA → CO2+ NADH + H+ + CH3-CO~SCoA
Connection between pyruvate dehydrogenase reaction and overall equation of the TCA cycle yields an equation that describes complete oxidation of pyruvate:
CH3-CO-COOH + 4NAD+ + FAD + ADP + Pi + 2H2O → 3CO2 + 4NADH + 4H+ + FADH2 + ATP
Individual reactions of the TCA cycle
Oxidation of acetyl residues includes several steps:
1) Acetyl residue (2C) is transferred to oxaloacetate (4C). This reaction is catalysed by the enzyme citrate synthase. Citrate (6C) is generated. This condensation reaction is irreversible, i.e. this is one of the regulatory steps of the TCA cycle.
2) Citrate is isomerised to isocitrate. Isocitrate is generated via aconitate using aconitate-hydratase (aconitase). This step is freely reversible.
3) Isocitrate is oxidised to α-ketoglutarate. This step is catalysed by the enzyme isocitrate dehydrogenase. This reaction is oxidative decarboxylation. This means (1) oxidation of –OH group to keto group (this yields NADH) and (2) one carboxylic group is broken apart (this yields CO2). This reaction is irreversible, i.e. this is one of the regulatory steps of the TCA cycle. This one is however the most important regulatory step of the TCA cycle.
4) α-ketoglutarate is oxidised to succinyl-CoA. This step is catalysed by the enzyme α-ketoglutarate dehydrogenase (multienzyme complex). This reaction is oxidative decarboxylation as well (thus yields CO2 and NADH). Reaction is irreversible, i.e. it is likewise the regulatory step.
5) Conversion of succinyl-CoA to succinate and co-enzym A. This step is catalysed by succinyl-CoA-ligase. This reaction is typical example of substrate fosforylation, i.e. the GTP is produced and is converted to the ATP. This reaction is reversible.
In reactions (1) to (5) acetyl residues is completely oxidised to 2 CO2, oxaloacetate is reduced to succinate. Oxaloacetate is regenerated from succinate during following reactions:
6) Succinate oxidation to fumarate is catalysed by succinate dehydrogenase. This enzyme is an integral protein in the inner mitochondrial membrane. It is part of the ETC – complex II. Succinate dehydrogenase uses FAD. This co-enzyme is reduced thus generating FADH2.
7) Water is added to double bond in fumarate and malate is produced. This step is catalysed by the enzyme fumarate hydratase (fumarase).
8) Malate is oxidised to oxaloacetate. This reaction is catalysed by the enzyme malate dehydrogenase. NADH is produced. This is the last step of the TCA cycle.
Products of the TCA cycle
In one turnover of the TCA cycle (i.e. one acetyl residue is processed) 2 CO2, 3 NADH, 1 FADH2 and 1 GTP are produced.
Carbon dioxide diffuses from mitochondria to blood. At the end CO2 is removed in the lungs. The reduced coenzymes (NADH and FADH2) are substrates for the ETC. The ETC produces the ATP. Overall energetic result of the TCA cycle is 10-12 ATP per one molecule AcCoA. In Subchapter 2/7 is described fact that it is not precise value and its evaluation is very problematic.
Replenishing (anaplerotic) reactions
Intermediates of the TCA cycle are in mitochondria in very low concentrations. Their constant regeneration takes place when acetyl residues are oxidised, hence their concentrations are held stable. On the other hand anabolic pathways drain intermediates from the TCA cycle. E.g. succinyl-CoA → hem synthesis, oxaloacetate → gluconeogenesis etc. Nevertheless intermediates could be replenished by so called anaplerotic reactions:
1) Pyruvate is carboxylated to oxaloacetate. This reaction is catalysed by the enzyme pyruvate carboxylase (cofactor is biotin – vitamin B7):
Pyruvate + CO2 + ATP → oxaloacetate + ATP + Pi
2) Carbon skeletons of amino acids are used to produce some intermediates of the TCA cycle (oxaloacetate, α-ketoglutarate, fumarate). For example aspartate could undergo transamination to oxaloacetate. Respectively glutamate to α-ketoglutarate.
3) Propionyl-CoA to succinyl-CoA. This is the way the odd-numbered chain fatty acid can enter the TCA cycle.
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Regulation of the TCA cycle
Regulatory steps of the TCA cycle:
1) Citratesynthase
2) Isocitratedehydrogenase
3) α-ketoglutaratedehydrogenase
Regulatory factors of the TCA cycle:
1) NADH/NAD+ ratio – respiratory control
2) ATP/ADP (AMP) ratio – energetic control
3) Availability of substrates for the TCA cycle – substrate control
1) NADH/NAD+ ratio – respiratory control
The TCA cycle continues to the ETC. Reoxidation of the reduced coenzymes takes place there. In situations when is the ETC slowed, NADH and FADH2 accumulate. It is obvious that NADH/NAD ratio increases and thus α-ketoglutaratedehydrogenase and isocitratedehydrogenase are inhibited.
2) ATP/ADP (AMP) ratio – energetic control
α-ketoglutaratedehydrogenase and isocitratedehydrogenase are inhibited when there is a sufficient amount of energy, i.e. ATP/ADP (AMP) ratio is high. ATP acts as inhibitor, ADP and AMP are activators of those two enzymes.
3) Availability of substrates for the TCA cycle – substrate control
As you know, velocity of the chemical reaction depends among others on the concentration of the reactants and the products. Velocity of the TCA cycle depends on the concentration of the citrate. Activity of the citratesynthase is related to amounts of oxaloacetate and AcCoA that are provided.
Activity of the TCA cycle is interwoven with the availability of the oxygen despite the fact that none of the reactions of the TCA cycle require oxygen. Oxygen is vital for the ETC. Oxygen is the final acceptor of the electrons. In the ETC reoxidation of the NADH → NAD+ and FADH2 → FAD take place. One can easily deduce that reduction of the oxygen supplies for the cell leads to drop of concentrations of the NAD+ and FAD, hence activity of the TCA cycle is decreased.
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Subchapter Author: Josef Fontana a Petra Lavríková
