Isocitrate, from citrate

Figure 7.49 Formation of isocitrate from citrate as catalyzed by the enzyme aconitase.

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. 

FIGURE 20.3 Martius and Knoop s observation that citrate could be converted to isocitrate aud then a-ketoglutarate provided a complete pathway from citrate to oxaloacetate. 

It may seem surprising that isocitrate dehydrogenase is strongly regulated, because it is not an apparent branch point within the TCA cycle. However, the citrate/isocitrate ratio controls the rate of production of cytosolic acetyl-CoA, because acetyl-CoA in the cytosol is derived from citrate exported from the mitochondrion. (Breakdown of cytosolic citrate produces oxaloacetate and acetyl-CoA, which can be used in a variety of biosynthetic processes.) Thus, isocitrate dehydrogenase activity in the mitochondrion favors catabolic TCA cycle activity over anabolic utilization of acetyl-CoA in the cytosol. 

Citrate is isomerized to isocitrate by the enzyme aconitase (aconitate hydratase) the reaction occurs in two steps dehydration to r-aconitate, some of which remains bound to the enzyme and rehydration to isocitrate. Although citrate is a symmetric molecule, aconitase reacts with citrate asymmetrically, so that the two carbon atoms that are lost in subsequent reactions of the cycle are not those that were added from acetyl-CoA. This asymmetric behavior is due to channeling— transfer of the product of citrate synthase directly onto the active site of aconitase without entering free solution. This provides integration of citric acid cycle activity and the provision of citrate in the cytosol as a source of acetyl-CoA for fatty acid synthesis. The poison fluo-roacetate is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits aconitase, causing citrate to accumulate. 

B. The rate-limiting step of the TCA cycle is the synthesis of a-ketoglutarate from citrate, catalyzed by isocitrate dehydrogenase (Figure 7—2). 

To understand why isocitrate dehydrogenase is so intensely regulated we must consider reactions beyond the TCA cycle, and indeed beyond the mitochondrion (fig. 13.15). Of the two compounds citrate and isocitrate, only citrate is transported across the barrier imposed by the mitochondrial membrane. Citrate that passes from the mitochondrion to the cytosol plays a major role in biosynthesis, both because of its immediate regulatory properties and because of the chain of covalent reactions it initiates. In the cytosol citrate undergoes a cleavage reaction in which acetyl-CoA is produced. The other cleavage product, oxaloacetate, can be utilized directly in various biosynthetic reactions or it can be converted to malate. The malate so formed can be returned to the mitochondrion, or it can be converted in the cytosol to pyruvate, which also results in the reduction of NADP+ to NADPH. The pyruvate is either utilized directly in biosynthetic processes, or like malate, can return to the mitochondrion. 

This differs from Figure 16-7 in that it does not include cis-aconitate and isocitrate (between citrate and a-ketoglutarate), or succinyl-CoA, or acetyl-CoA. 

A total of 14 NADPH molecules are utilized to make each palmitate molecule. It comes from three sources the malic enzyme (see earlier) provides one NADPH molecule for every acetyl-CoA molecule generated from citrate. For palmitate, this accounts for eight NADPH molecules. The rest must be derived largely from the hexose monophosphate shunt (see Chapter 18). A minor source of NADPH is cytosolic isocitrate dehydrogenase (see Chapter 18). The synthesis of one palmitate molecule thus requires an equivalent of 7 + (3)14 = 49 ATP molecules. 

Aconitase catalyzes the removal of water from citrate to form c/.s-aconitate, and water is added to c/.v-aconitate to form either citrate again or d-isocitrate, depending on whether the hydroxyl is on the third or second carbon. 

Cis-aconitate is transiently formed as a product bound to the enzyme. The compound is formed from citrate by removal of a water, which is added back to obtain isocitrate (reaction diagram). 

Figure 6 Catalytic mechanism of aconitase starting from citrate (left) or isocitrate (right).

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. 

Figure 7 Ion exclusion isocratic separation of organic acids. Peaks 1 chloride, 2 oxalate, 3 pyruvate, 4 tartrate, 5 malonate, 6 lactate, 7 malate, 8 acetate, 9 isocitrate, 10 citrate, 11 p-hydroxy-n-butyrate, 12 succinate, 13 proprionate. (Reproduced with permission from Dionex Product Selection Guide 1991.)...

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. 

AMP is an activator of the enzyme, isocitrate dehydrogenase (ICDH), found in the mitochondrion. ICDH catalyses the metabolism of isocitrate (reaction (9.2)) which in turn is derived immediately from citrate (reaction (9.3)). Citrate itself is a direct product of glucose metabolism (Figure 9.4). 

Oxaloacetate can be regenerated from both cleavage products of isocitrate From glyoxylate by condensation with acetyl-CoA to form malate which is subsequently dehydrogenated, and from succinate through the usual citrate cycle. As a net result, 2 moles of activated acetate have been converted to succinate which can undergo further reactions by familiar pathwa3rs. Other synthetic pathways branch of these secondary products. 

FIGURE 20.7 (a) The aconitase reaction converts citrate to cis-aconitate and then to isocitrate. Aconitase is stereospecific and removes the pro-/ hydrogen from the pro-/ arm of citrate, (b) The active site of aconitase. The iron-sulfur cluster (red) is coordinated by cysteines (yellow) and isocitrate (white). 

Step 2 of Figure 29.12 Isomerization Citrate, a prochiral tertiary alcohol, is next converted into its isomer, (2, 35)-isocitrate, a chiral secondary alcohol. The isomerization occurs in two steps, both of which are catalyzed by the same aconitase enzyme. The initial step is an ElcB dehydration of a /3-hydroxy acid to give cfs-aconitate, the same sort of reaction that occurs in step 9 of glycolysis (Figure 29.7). The second step is a conjugate nucleophilic addition of water to the C=C bond (Section 19.13). The dehydration of citrate takes place specifically on the pro-R arm—the one derived from oxaloacetate—rather than on the pro-S arm derived from acetyl CoA. 

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 ...

Figure 3 Gradient separation of anions using suppressed conductivity detection. Column 0.4 x 15 cm AS5A, 5 p latex-coated resin (Dionex). Eluent 750 pM NaOH, 0-5 min., then to 85 mM NaOH in 30 min. Flow 1 ml/min. 1 fluoride, 2 a-hydrox-ybutyrate, 3 acetate, 4 glycolate, 5 butyrate, 6 gluconate, 7 a-hydroxyvalerate, 8 formate, 9 valerate, 10 pyruvate, 11 monochloroacetate, 12 bromate, 13 chloride, 14 galacturonate, 15 nitrite, 16 glucuronate, 17 dichloroacetate, 18 trifluoroacetate, 19 phosphite, 20 selenite, 21 bromide, 22 nitrate, 23 sulfate, 24 oxalate, 25 selenate, 26 a-ketoglutarate, 27 fumarate, 28 phthalate, 29 oxalacetate, 30 phosphate, 31 arsenate, 32 chromate, 33 citrate, 34 isocitrate, 35 ds-aconitate, 36 trans-aconitate. (Reproduced with permission of Elsevier Science from Rocklin, R. D., Pohl, C. A., and Schibler, J. A., /. Chromatogr., 411, 107, 1987.)...

Figure 9 A synthetic mixture of water-soluble carboxylic acids separated by anion-exchange chromatography. Column 0.3 cm x 300 cm Diaoion CA 08, 16-20 p (Mitsubishi Kasei Kogyo). Eluant 200 mM HC1. Detection reaction with Fe3-benzohy-droxamic acid-dicyclohexy carbodiimide-hydroxylamine perchlorate-triethyl amine with absorbance at 536 nm. Analytes (1) aspartate, (2) gluconate, (3) glucuronate, (4) pyroglutamate, (5) lactate, (6) acetate, (7) tartrate, (8) malate, (9) citrate, (10) succinate, (11) isocitrate, (12) w-butyrate, (13) a-ketoglutarate. (Reprinted with permission from Kasai, Y., Tanimura, T., and Tamura, Z., Anal. Chem., 49, 655, 1977. 1977 Analytical Chemistry).

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... 

This interpretation offered by Ogston was confirmed by further isotope experiments (Potter and Heidelberger, 1949 Martius and Schorre, 1950). Ochoa and his colleagues also demonstrated that in pigeon-liver preparations which had been freed from aconitase, thus preventing isomerization between citrate and isocitrate, citrate was the product of the condensation of oxaloacetate and acetate. 

---