Citrate tricarboxylic acid cycl

The citrate cycle is the final common pathway for the oxidation of acetyl-CoA derived from the metabolism of pyruvate, fatty acids, ketone bodies, and amino acids (Krebs, 1943 Greville, 1968). This is sometimes known as the Krebs or tricarboxylic acid cycle. Acetyl-CoA combines with oxaloacetate to form citrate which then undergoes a series of reactions involving the loss of two molecules of CO2 and four dehydrogenation steps. These reactions complete the cycle by regenerating oxaloacetate which can react with another molecule of acetyl-CoA (Figure 4). 

Wachtcrshauser s prime candidate for a carbon-fixing process driven by pyrite formation is the reductive citrate cycle (RCC) mentioned above. Expressed simply, the RCC is the reversal of the normal Krebs cycle (tricarboxylic acid cycle TCA cycle), which is referred to as the turntable of metabolism because of its vital importance for metabolism in living cells. The Krebs cycle, in simplified form, can be summarized as follows ... 

Sherwood-Jones et al.2 have demonstrated the presence of the tricarboxylic acid cycle in mammalian reticulocytes by observing citrate accumulation in the presence of sodium fluoroacetate. They also demonstrated a substantial inhibition of respiration by fluorocitrate. 

The tricarboxylic acid cycle was therefore validated, having been tested not only in pigeon-breast muscle but also with brain, testis, liver, and kidney. The nature of the carbohydrate fragment entering the cycle was still uncertain. The possibility that pyruvate and oxaloacetate condensed to give a 7C derivative which would be decarboxy-lated to citrate, was dismissed partly because the postulated compound was oxidized at a very low rate. Further, work on the oxidation of fatty acids (see Chapter 7) had already established that a 2C fragment like acetate was produced by fatty acid oxidation, en route for carbon dioxide and water. It therefore seemed likely that a similar 2C compound might arise by decarboxylation of pyruvate, and thus condense with oxaloacetate. For some considerable time articles and textbooks referred to this unknown 2C compound as active acetate. ... 

The consequent interpretation, accepted by Krebs in his review of the tricarboxylic acid cycle in 1943, was therefore that citric acid could not be an intermediate on the main path of the cycle, and that the product of the condensation between oxaloacetate and acetyl CoA would have to be isocitrate, which is asymmetric. This view prevailed between 1941 and 1948 when Ogston made the important suggestion that the embarrassment of the asymmetric treatment of citrate could be avoided if the acid was metabolized asymmetrically by the relevant enzymes, citrate synthase and aconitase. If the substrate was in contact with its enzyme at three or more positions a chiral center could be introduced. 

The toxicity of the acaricide 2-fluoro-A-methyl-A-(naphth-l-yl)acet-amide (MNFA) (4.152) to mammals is related to its hydrolysis. In this case, however, toxicity was mainly due to the acid formed. Indeed, the 2-fluoro-acetic acid (4.153) liberated by hydrolysis was further metabolized to fluoro-citrate, which inhibits the tricarboxylic acid cycle [101]. 

The tricarboxylic acid cycle not only takes up acetyl CoA from fatty acid degradation, but also supplies the material for the biosynthesis of fatty acids and isoprenoids. Acetyl CoA, which is formed in the matrix space of mitochondria by pyruvate dehydrogenase (see p. 134), is not capable of passing through the inner mitochondrial membrane. The acetyl residue is therefore condensed with oxaloacetate by mitochondrial citrate synthase to form citrate. This then leaves the mitochondria by antiport with malate (right see p. 212). In the cytoplasm, it is cleaved again by ATP-dependent citrate lyase [4] into acetyl-CoA and oxaloacetate. The oxaloacetate formed is reduced by a cytoplasmic malate dehydrogenase to malate [2], which then returns to the mitochondrion via the antiport already mentioned. Alternatively, the malate can be oxidized by malic enzyme" [5], with decarboxylation, to pyruvate. The NADPH+H formed in this process is also used for fatty acid biosynthesis. 

Fluoroacetate causes inhibition of aconitase, an enzyme in the tricarboxylic acid cycle. This is due to the formation of fluorocitrate, which binds to aconitase and inhibits the enzyme. This is because the fluorine atom cannot be removed from the fluorocitrate unlike the hydrogen atom in the normal substrate, citrate. The result is complete blockade of the cycle and this means tissues become starved of ATP and other vital metabolic intermediates. This causes adverse effects in the heart as the organ is particularly sensitive to deficiency of ATP. 

Acetyl CoA is produced in the mitochondria and condenses with OAA to form citrate, the first step in the tricarboxylic acid cycle. 

Notice in the initial steps of the tricarboxylic acid cycle, citrate is converted to isocitrate, which is then oxidized. Why didn t nature just oxidize citrate and save an enzymatic step ... 

Notice the intermediate in the reaction of citrate synthase (fig. 13.7). Do you think at some time in the future evolution will produce a variety of citrate synthase that recovers the energy in the thioester, analogous to the production of GTP (ATP) by succinate thiokinase (page 291) Would this energy recovery have any effect on the thermodynamics of the tricarboxylic acid cycle ... 

The availability of citrate has no relationship to the flow of metabolites through the tricarboxylic acid cycle. Increased citrate concentrations result in increased cytoplasmic acetyl-CoA concentrations, which in turn increases fatty acid biosynthesis. 

R. Bentley, A history of the reaction between oxaloacetate and acetate for citrate biosynthesis an unsung contribution to the tricarboxylic acid cycle , Perspect. Biol. Med., 1994, 37, 362-383. 

The effect of nonfatal injuries such as a 2-hour period of bilateral hind-limb ischemia or a full-thickness scald of 20% of skin surface on the LDso of DNOC and its hyperthermic effect were evaluated in male rats (Stoner 1969). The intraperitoneal LDs° of DNOC was significantly (p<0.001) reduced from 24.8 to 26.2 mg/kg to 14 mg/kg DNOC when DNOC was given 1.5- 24 hours after either type of nonfatal injury. The authors concluded that the toxicity of DNOC was increased by previous trauma. These investigators proposed that this interaction was associated with sequential blocking of the tricarboxylic acid cycle with inhibition of citrate synthetase reaction during the early part of the response to the injury. Because DNOC acts as an uncoupler of oxidative phosphorylation, less ATP is produced. Therefore, the effects of trauma will be enhanced by an uncoupling agent such as DNOC. 

This finding ruled out citrate as a direct intermediate in the tricarboxylic acid cycle since isotopic citric acid, which is a symmetrical compound, could only give rise to a-ketoglutaric acid containing isotope distributed equally in both carboxyl groups (Fig. 5). Addition of citrate to their system (Evans and Slotin), furthermore, did not affect the specific radioactivity of the o-ketoglutarate. This effect would have been obtained had citrate been an intermediate. 

The first reaction of the tricarboxylic acid cycle, the citrate s)mthesis... 

The activity of an enzyme may be inhibited by the presence of a toxic metabolite. Sodium fluoroacetate, known as rat poison 1080, is extremely toxic to animals. The toxic action, however, is not due to sodium fluoroacetate itself but to a metabolic conversion product, flu-orocitrate, formed through a reaction commonly known as "lethal synthesis," as shown in Figure 5.3. The resulting fluorocitrate is toxic because it is inhibitory to aconitase, the enzyme responsible for the conversion of citrate into czs-aconitate and then into isocitrate in the tricarboxylic acid cycle. Inhibition of aconitase results in citrate accumulation. The cycle stops for lack of metabolites, leading to disruption of energy metabolism. 

The second metabolic pathway which we have chosen to describe is the tricarboxylic acid cycle, often referred to as the Krebs cycle. This represents the biochemical hub of intermediary metabolism, not only in the oxidative catabolism of carbohydrates, lipids, and amino acids in aerobic eukaryotes and prokaryotes, but also as a source of numerous biosynthetic precursors. Pyruvate, formed in the cytosol by glycolysis, is transported into the matrix of the mitochondria where it is converted to acetyl CoA by the multi-enzyme complex, pyruvate dehydrogenase. Acetyl CoA is also produced by the mitochondrial S-oxidation of fatty acids and by the oxidative metabolism of a number of amino acids. The first reaction of the cycle (Figure 5.12) involves the condensation of acetyl Co and oxaloacetate to form citrate (1), a Claisen ester condensation. Citrate is then converted to the more easily oxidised secondary alcohol, isocitrate (2), by the iron-sulfur centre of the enzyme aconitase (described in Chapter 13). This reaction involves successive dehydration of citrate, producing enzyme-bound cis-aconitate, followed by rehydration, to give isocitrate. In this reaction, the enzyme distinguishes between the two external carboxyl groups... 

Interference with the first stage of the tricarboxylic acid cycle should by deprivation, lead to a fall in the concentration of compounds such as citrate, glutamate, and aspartate and also to the accumulation of pyruvate. These changes have been observed in the liver of the injured rat. Liver mitochondria isolated from injured rats, even from those on the point of death still show normal behavior in vitro (A2). 

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