Conversion of isocitrate

As mentioned in the introductory part, stereochemical course of the conversion of isocitric acid to a-ketoglutaric acid in TCA cycle is completely enantiose-lective although the reaction does not form an asymmetric carbon in the usual metabolic path. If such type of oxidative decarboxylation can be applied to synthetic compounds, it is expected that an entirely new type of asymmetric biotransformation will be developed. 

This [4Fe-4S] cluster-containing enzyme [EC 4.2.1.3], also known as citrate hydro-lyase and aconitate hydra-tase, will act on citrate to generate ds-aconitate ((Z)-prop-l-ene 1,2,3-tricarboxylate) and water. The enzyme will also catalyze the conversion of isocitrate into ds-aconitate. 

This enzyme [EC 4.1.3.1] (also referred to as isocitrase, isocitritase, and isocitratase) catalyzes the conversion of isocitrate (specifically, (li ,25)-l-hydroxypropane 1,2,3-tricarboxylate) to succinate and glyoxylate. 

In the conversion of isocitrate to a-ketoglutarate, oxidation and decarboxylation steps occur. Figure 13.9 shows the oxidation step first. Can you suggest a reason why the oxidation step is first ... 

Isocitrate Dehydrogenase Reaction What type of chemical reaction is involved in the conversion of isocitrate to a-ketoglutarate Name and describe the role of any cofactors. What other reaction(s) of the citric acid cycle are of this same type ... 

The conversion of isocitrate into a-ketoglutarate is followed by a second oxidative decarboxylation reaction, the formation of succinyl CoA from a-ketoglutarate. 

B. In the conversion of isocitrate (Compound A) to fumarate (Compound B), 2 C02, NADH (which contains niacin), 1 GTP, and 1 FADH2 are produced. A total of approximately 9 ATP are generated. The enzymes for these reactions are all located in the mitochondrial matrix except succinate dehydrogenase, which is in the inner mitochondrial membrane. GTP does not drive any of the reactions. 

Immediate decarboxylation of oxalosuccinate results in the formation of a-keto-glu-tarate, an a-keto acid. There are two forms of isocitrate dehydrogenase in mammals. The NAD+-requiring isozyme is found only within mitochondria. The other isozyme, which requires NADP+, is found in both the mitochondrial matrix and the cytoplasm. In some circumstances the latter enzyme is used within both compartments to generate NADPH, which is required in biosynthetic processes. Note that the NADH produced in the conversion of isocitrate to a-ketoglutarate is the first link between the citric acid cycle and the ETC and oxidative phosphorylation. 

Conversion of pyruvate to acetyl-CoA, conversion of isocitrate to a-ketogluta-rate, and conversion of a-ketoglutarate to succinyl-CoA. 

The conversion of isocitrate to succinate and glyoxylate catalyzed by isocitrate lyase and the conversion of glyoxylate and acetyl-CoA to malate catalyzed by malate synthase. 

Examples of reactions generating NADH include the conversion of isocitrate into ketoglutarate, and malate into oxaloacetate in the Krebs cycle (Figure 11.21), the oxidation of fatty acids (11.96), the conversion of glyceraldehyde-3-phosphate into 1,3-diphosphoglycerate in glycolysis (Figure 11.20), and reaction... 

Plants, hut not animals, are able to synthesize glucose from acetyl CoA by a pathway that begins with the glyoxalate cycle. One of the steps in the cycle is the conversion of isocitrate to glyoxalate plus succinate, a process catalyzed by isocitrate lyase. Propose a mechanism for the reaction. 

Formation of phosphocreatine (Jacobs et al., 1964) Conversion of isocitrate to 2-oxoglutarate (Henderson, 1965) Formation of glucose 6-phosphate (Niemeyer et al., 1975)... 

Several mutant strains of R. eutropha that were made to possess defective competing metabolic pathways with the PHA biosynthetic pathway were developed for the enhanced PHA production. The isocitrate dehydrogenase leaky mutant of R. eutropha accumulated P(3HB) more favorably at a lower car-bon/nitrogen molar ratio and at a lower carbon concentration than the parent strain [82]. In batch culture, the final cell and P(3HB) concentrations, and P(3HB) yield on glucose were slightly increased. Also, in the P(3HB-co-3HV) biosynthesis, the molar fraction of 3HV and the 3HV yield on propionic acid increased due to the enhanced conversion of propionic acid to 3-hydroxyvaleryl-CoA rather than to acetyl-CoA and C02 in this mutant. Another mutant R. eu-... 

Aconitase, an unstable enzyme,4 is concerned with the reversible conversion of cis-aconitate to either citric acid or isocitric acid. It may be noted that the entire system of tricarboxylic cycle enzymes are present in the mitochondria separated from cells, and, furthermore, it has been found that the mitochondrial enzymes differ from the isolated enzymes in that the former require no addition of D.P.N. (co-enzyme I) or T.P.N. (co-enzyme II) for activity. Peters suggests that the citrate accumulation is caused by the competitive reaction of the fluorocitrate with aconitase required for the conversion of citrate to isocitrate. This interference with the tricarboxylic acid... 

The catalytic efficiency of this enzyme to hydrolyze 5-fluoro-5,6-dihydro-uracil was found to be approximately twice that toward 5,6-dihydrouracil [152], 2-Fluoro-/3-alanine can either be eliminated via the bile after conjugation with bile acids, or be converted to fluoroacetate (4.238) [153], The latter metabolite is transformed to fluorocitrate, a potent inhibitor of the aconi-tase-catalyzed conversion of citrate to isocitrate. This inhibition probably explains the clinical neurotoxicity of 5-fluorouracil [154] [155],... 

Enzymes usually function stereospedfically. In chiral substrates, they only accept one of the enantiomers, and the reaction products are usually also sterically uniform. Aconitate hydratase (aconitase) catalyzes the conversion of citric acid into the constitution isomer isocitric acid (see p.l36). Although citric acid is not chiral, aconitase only forms one of the four possible isomeric forms of isocitric acid (2i ,3S-isocitric acid). The intermediate of the reaction, the unsaturated tricarboxylic acid aconitate, only occurs in the cis form in the reaction. The trans form of aconitate is found as a constituent of certain plants. 

Fluoroacetate produces its toxic action by inhibiting the citric acid cycle. The fluorine-substituted acetate is metabolized to fluoroci-trate that inhibits the conversion of citrate to isocitrate. There is an accumulation of large quantities of citrate in the tissue, and the cycle is blocked. The heart and central nervous system are the most critical tissues involved in poisoning by a general inhibition of oxidative energy metabolism. ... 

The overall rate of the citric acid cycle is controlled by the rate of conversion of pyruvate to acetyl-CoA and by the flux through citrate synthase, isocitrate dehydrogenase, and a-lcetoglutarate dehydrogenase. These fluxes are largely determined by the concentrations of substrates and products the end products ATP and NADH are inhibitory, and the substrates NAD+ and ADP are stimulatory. 

The first oxidative conversion of the TCA cycle is catalyzed by isocitrate dehydrogenase. This conversion takes place in two steps oxidation of the secondary alcohol to a ketone (oxalosuccinate), followed by a j8 decarboxylation to produce a-ketoglutarate (fig. 13.9). 

The equilibrium constant for the conversion of citrate to isocitrate is small, and the interconversion is rapid, so these... 

While the mannitol cycle is generating NADPH, another cycle, involving isocitrate and 2-ketoglutarate, may operate in the opposite direction [53] (Fig. 10). The net reaction is the conversion of NADPH and NAD+ into NADP+ and NADH, respectively. Using gas chromatography/mass spectrometry, evidence for the activity of the cycle was indicated by the appearance of citrate enriched in five positions, when U-UC5 glutamate was being metabolized in the perfused rat liver. 

Both of these reactions are followed by exergonic reactions. The equilibrium of the reaction malate oxaloacetate (step 8) lies in favor of malate formation, so at equilibrium the concentration of oxaloacetate will be low. The next reaction in the cycle (oxaloacetate + acetyl-CoA — citrate) (step 1) is, however, exergonic, and the oxaloacetate is removed to condense with acetyl-CoA. Similarly, the conversion of citrate to isocitrate is endergonic, and at equilibrium the reaction favors the formation of citrate. The next reaction in the cycle (isocitrate—>2-oxoglutarate) is exergonic, and so the isocitrate is removed thus allowing this reaction to proceed. 

---