Isocitrate, from citrate oxidation

Another important piece of the puzzle came from the work of Carl Martius and Franz Knoop, who showed that citric acid could be converted to isocitrate and then to a-ketoglutarate. This finding was significant because it was already known that a-ketoglutarate could be enzymatically oxidized to succinate. At this juncture, the pathway from citrate to oxaloacetate seemed to be as shown in Figure 20.3. Whereas the pathway made sense, the catalytic effect of succinate and the other dicarboxylic acids from Szent-Gyorgyi s studies remained a puzzle. 

Citrate that is not oxidized by isocitrate dehydrogenase can be transported from the mitochondrial matrix into the cytoplasm. In the cytoplasm of adipocytes and hepatocytes, oxaloacetate and acetyl-CoA are formed from citrate, not by the reversal of the citrate synthase-catalyzed reaction, bnt by ATP-dependent citrate lyase. As the name indicates, the free energy of ATP hydrolysis drives this reaction in the degradative direction. 

IsocHricacid HOOC-CH2-CH(COOH)-CHOH-COOH, a monohydroxy tricarboxylic acid, an isomer of citric acid, which is widely distributed in the plant kingdom and occurs in free form especially in plants of the stone-crop family (Crassulaceae), and in fruits. The salts of I. a., isocitrates, are important metaboli-cally as intermediates in the Tricarboxylic acid cycle (see), where they are formed from citrate by the enzyme aconitase, then oxidized to 2-oxoglutarate. In the Glyoxylate cycle (see), isocitrate is cleaved to succinate and glyoxylate. 

Figure 4. The citrate cycle. There is complete oxidation of one molecule of acetyl-CoA for each turn of the cycle CH3COSC0A + 2O2 - 2CO2 + H2O + CoASH. The rate of the citrate cycle is determined by many factors including the ADP/ATP ratio, NAD7NADH ratio, and substrate concentrations. During muscle contraction, Ca is released from cellular stores (mainly the sarcoplasmic reticulum) and then taken up in part by the mitochondria (see Table 2). Ca " activates 2-oxoglutarate and isocitrate dehydrogenases (Brown, 1992). Succinate dehydrogenase may be effectively irreversible. Enzymes ...

Perhaps the best characterized example of a subsite differentiated [4Fe-4S] protein is aconitase, which catalyzes the citrate-isocitrate isomerization in the citric acid cycle (257). Aconitase isolated aerobically is inactive and contains a [3Fe-4S] cluster. Activity is restored by incubation with Fe and this also reconstitutes the [4Fe-4S] cluster. Oxidation of the core results in loss of the fourth iron atom, regenerating the [3Fe-4S] form. Mossbauer studies have demonstrated that only one of the four iron sites is exchanged (258). X-ray studies on both [3Fe-4S] and [4Fe-4S] forms of pig heart aconitase 258a) showed that insertion of iron into [3Fe-4S] occurs isomorphously. The positions of the common atoms in the two forms of the core agree to within 0.1 A, supporting the view of the [3Fe-4S] cluster as an iron-voided cubane. A similar result was obtained for the seven iron ferredoxin from Azo-... 

Aconitase catalyzes the isomerization of citrate to isodtrate, isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to a-ketoglutarate, and a-ketoglutarate dehydrogenase catalyzes the oxidative decarboxylation of a-keto-glutarate to succinyl-CoA. Succinyl-CoA and the remaining intermediates are the 4-carbon intermediates of the Krebs cycle. Succinyl thiokinase catalyzes the release of coenzyme A from succinyl-CoA and the production of GTP. Succinate dehydro-... 

Note that the dehydration of citrate takes place specifically away from the carbon atoms of the acetyl group that added to oxaloacetate in step 1. steps 3-4 Oxidative decarboxylations. Isocitrate, a secondary alcohol, is oxidized by NAD in step 3 to give a ketone, which loses C02 to give -ketoglutarate. Catalyzed by the enzyme isocitrate dehydrogenase, the decarboxylation is a typical reaction of a 0-keto acid, just like that in the acetoacetic ester synthesis (Section 22.8). 

Rapid p-oxidation of fatty acids in perfused liver (DeBeer et a/., 1974) and in isolated mitochondria (Lopes-Cardozo and Van den Bergh, 1972) has been shown to suppress the operation of citric acid cycle apparently from the elevation of mitochondrial NADH/NAD ratio which restricts oxaloaceta-te availability for citrate synthase and simultaneously inhibits isocitrate oxidation (Lenartowicz et a/., 1976). Considerable support for an earlier postulate that oxaloacetate availability normally determines the rate of citrate synthesis has become available. Thus, because of marked protein binding, the concentration of free, as opposed to total, oxaloacetate in matrix of liver mitochondria is now estimated to be near the of citrate synthase (Siess et al., 1976 Brocks eta ., 1980). The antiketogenic effect of alanine (Nosadini et a/., 1980) and of 3-mercaptopicolinate, an inhibitor of phosphoenolpy-ruvate carboxykinase (Blackshear et a/., 1975), is believed to be exerted, at least in part, from their ability to raise hepatic oxaloacetate concentration. And, in pyruvate carboxylase deficiency, expected to impair oxaloacetate supply, concentration of ketone bodies is elevated (Saudubray et a/., 1976). 

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