Krebs identified the citric acid cycle in 1937 by noting that small
quantities of organic acids such as succinate, malate or citrate stimulated
oxygen uptake by minced pigeon breast muscle. He realised that the amount
of additional oxygen consumed was greater than that required for the complete oxidation of the added material, and deduced that the acids must act catalytically.
Most of the oxygen was consumed by the endogenous substrates that were already present in the mince. Using inhibitors (such as malonate, which blocks succinate oxidation) he showed that the supplementary organic acids participated in the oxidative processes, but they were normally regenerated by the tissue metabolism. Although several key intermediates and cofactors remained to be discovered, there was only one plausible sequence for the known participants and the cycle was established.
"Nature" rejected Krebs' paper as insufficiently important, and he was obliged to publish his results in "Experientia" instead.
The development of faster centrifuges in the late 1940's permitted the isolation of subcellular organelles such as nuclei and mitochondria. It was realised that the Krebs cycle took place within this particulate fraction, and that the enzyme activies were "latent" until the surrounding membranes were disrupted by mechanical forces or osmotic shock.
The detailed chemical structures have very limited medical significance, but you will find it very much easier to make sense of the other material in this course if you take the trouble to learn them! It may be helpful to follow one particular atom in acetyl CoA all the way round the cycle until it is lost as carbon dioxide, and the coloured boxes are intended to assist this process.
The Krebs cycle enzymes are soluble proteins located in the mitochondrial matrix space, except for succinate dehydrogenase, which is an integral membrane protein that is firmly attached to the inner surface of the inner mitochondrial membrane, where it communicates directly with components in the respiratory chain. Succinate dehydrogenase uses FAD as a prosthetic group, but the other three oxidation steps use NAD as their coenzyme. Most of the energy is captured when the reduced coenzymes are re-oxidised by the respiratory chain in the mitochondrial inner membrane, but there is a single "substrate level" phosphorylation catalysed by succinate thiokinase, which rather unusually uses GDP as its phosphate acceptor. We do not properly understand the reason for this. Despite the claims in some text books, the GTP may not communicate freely with other nucleotide pools.
The four dehydrogenase reactions differ considerably in their energy yield. Succinate has the lowest redox potential (the worst reducing agent) so its oxidation yields relatively little energy. Reoxidation of the FAD2H cofactor for succinate dehydrogenase by the respiratory chain will only support the formation of 1.5 ATP / mole. Malate is a better reducing agent, and will generate 2.5 ATP / mole when the NADH from malate dehydrogenase is re-cycled to NAD by the respiratory chain. Oxoglutatarate and especially isocitrate are even better reducing agents. They are restricted to 2.5 ATP / mole when they use NADH as their initial hydrogen carrier, but cells find it necessary to regulate these highly favourable reactions as described below.
Each turn of the cycle achieves the complete oxidation of one molecule of acetyl CoA to form 2 molecules of carbon dioxide. The process also yields 3 molecules of NADH, 1 of FAD2H and one molecule of high-energy phosphate in the form of GTP. Reoxidation of all the reduced coenzymes by the respiratory chain yields a further 9 ATP, so the total energy captured by the cycle is 10 ATP equivalents per mole of acetyl CoA. This represents an overall energy conversion efficiency of about 62 % [free energy change for the complete oxidation of acetic acid = 805 kJ/mole, free energy of hydrolysis of ATP = 50 kJ/mole under typical intracellular conditions].
The various intermediates are present at very different concentrations. Citrate, oxoglutarate, succinate and malate are major metabolites, and are typically present at concentrations approaching 1 mM in the matrix space. Isocitrate is about 5% of the citrate concentration, and fumarate about 25% of malate, fixed by enzyme equilibrium constants. Coenzyme A and its various derivatives are fifty times lower than this, and oxaloacetate is present in vanishingly small amounts, especially in the fasting state. Lack of oxaloacetate is a major constraint on citrate synthase activity.
Krebs cycle enzymes redistribute material between the various pools, but they do not change the total amount of substrate in circulation. Acetyl CoA, for example, is oxidised via the cycle, but its incorporation into citrate merely "robs Peter to pay Paul" and it does not change the total quantity of cycle intermediates. The availability of the four-, five- and six-carbon organic acids affects the activity of the cycle in vivo, just as Krebs observed in his original in vitro experiments in 1937.
Krebs cycle intermediate concentrations depend on the activity of ancillary enzymes, which add material to the cycle from amino acid or carbohydrate sources, or alternatively remove intermediates for use in biosynthetic reactions. An important anaplerotic (or "filling up") reaction is catalysed by pyruvate carboxylase, which forms oxaloacetate from pyruvate within the mitochondria and is powerfully activated by acetyl CoA. The degradation of most amino acids also adds considerably to the total pool of cycle intermediates. The most important emptying processes are the removal of malate and related compounds for the synthesis of carbohydrates, and the export of citrate from the mitochondria for the biosynthesis of fat.
Another major factor affecting Krebs cycle activity is the availability of cofactors such as acetyl CoA, CoASH, NAD, FAD and GDP. Since the reactions form a closed loop, all five requirements must be simultaneously satisfied before the complete cycle can proceed. There is competition between the various oxidative pathways for access to the mitochondrial respiratory chain, so that it is possible, for example, for fatty acid oxidation to proceed at high speed (leading to the formation of ketone bodies) while the Krebs cycle is almost at a standstill.
Two allosteric enzymes help to control the distribution of metabolites
and the overall cycle flux. The NAD-linked isocitrate dehydrogenase is
inhibited by ATP, NADH & NADPH, and activated by ADP & calcium
ions. Oxoglutarate dehydrogenase is also activated by calcium. Segments
of the cycle may still be active even when the complete cycle cannot take
place. This allows the cycle to perform its "clearing house" functions
independently of respiratory activity and oxidative phosphorylation.
Energy stores and inter-conversions in humans
Notes: (1) These figures are for a 70kg male. The average daily energy intake is about 12 MJ per day for males, 9.2 MJ for females, so the total stores would last about 40 days, providing water was available and blood glucose could be maintained through gluconeogenesis. In practice food withdrawal may not be complete, and reduced physical activity lowers fasting energy requirements. Human beings have evolved to withstand a bad winter in a primitive hunter-gatherer society.
(2) There is no net synthesis of amino acids under physiological conditions, but in the case of the non-essential amino acids it may be possible to use transamination "to rob Peter to pay Paul".
isocitrate dehydrogenase 3 (NAD, mitochondrial)
malate dehydrogenase (mitochondrial)
methylmalonyl CoA mutase
methylmalonyl CoA racemase
propionyl CoA carboxylase
If you have comments, queries or suggestions, email me at J.A.Illingworth@leeds.ac.uk