Isocitrate

Iron Sulfur Compounds. Many molecular compounds (18—20) are known in which iron is tetrahedraHy coordinated by a combination of thiolate and sulfide donors. Of the 10 or more stmcturaHy characterized classes of Fe—S compounds, the four shown in Figure 1 are known to occur in proteins. The mononuclear iron site REPLACE occurs in the one-iron bacterial electron-transfer protein mbredoxin. The [2Fe—2S] (10) and [4Fe—4S] (12) cubane stmctures are found in the 2-, 4-, and 8-iron ferredoxins, which are also electron-transfer proteins. The [3Fe—4S] voided cubane stmcture (11) has been found in some ferredoxins and in the inactive form of aconitase, the enzyme which catalyzes the stereospecific hydration—rehydration of citrate to isocitrate in the Krebs cycle. In addition, enzymes are known that contain either other types of iron sulfur clusters or iron sulfur clusters that include other metals. Examples include nitrogenase, which reduces N2 to NH at a MoFe Sg homocitrate cluster carbon monoxide dehydrogenase, which assembles acetyl-coenzyme A (acetyl-CoA) at a FeNiS site and hydrogenases, which catalyze the reversible reduction of protons to hydrogen gas. 

We detenuined the influence of oxy- and ketocarboxylic acids (succinate, fumarate, adipinate, a-ketoglutarate, isocitrate, tartrate, E-malate) on the luminescence intensity of the Eu-OxTc complex. These substances interact as polydentate ligands similarly to citrate with the formation of ternary complexes with Eu-OxTc. As to succinate, fumarate, adipinate and a-ketoglutarate this they cannot effectively coordinate with EiT+ and significant fluorescence enhancement was not observed. 

Assign configurations, using the sequence rule, to each chiral center of the stereo-isomeric isocitric acids and alloisocitric acids ... 

The enzyme aconitase catalyzes the hydration of aconitic acid to two products citric acid and isocitric acid. Isocitric acid is optically active citric acid is not. What are the respective constitutions of citric acid and isocitric acid ... 

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. 

AceCyl-CoA + oxaloacetate + HgO. CoASH + citrate 2. Citrate. isocitrate 3. Isocitrate + NAD. a-ketoglntarate + NADH + CO, + 4. a-Ketoglntarate + CoASH + NAD. snccinyl-CoA + NADH + CO, + H Citrate synthase Aconitase Isocitrate dehydrogenase u-Ketoglutarate dehydrogenase complex... 

Citrate is isomerized to isocitrate by aconitase in a two-step process involving aconitate as an intermediate (Figure 20.7). In this reaction, the elements... 

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). 

Isocitrate Dehydrogenase Links the TCA Cycle and Electron Transport... 

FIGURE 20.10 (a) The isocitrate dehydrogenase reaction, (b) The active site of isocitrate dehydrogenase. Isocitrate is shown in green, NADP is shown in gold, with Ca" in red. 

One of these alternate models, postulated by Gunter Wachtershanser, involves an archaic version of the TCA cycle running in the reverse (reductive) direction. Reversal of the TCA cycle results in assimilation of CO9 and fixation of carbon as shown. For each turn of the reversed cycle, two carbons are fixed in the formation of isocitrate and two more are fixed in the reductive transformation of acetyl-CoA to oxaloacetate. Thus, for every succinate that enters the reversed cycle, two succinates are returned, making the cycle highly antocatalytic. Because TCA cycle intermediates are involved in many biosynthetic pathways (see Section 20.13), a reversed TCA cycle would be a bountiful and broad source of metabolic substrates. 

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. 

Isocitrate Lyase Short-Circuits the TCA Cycle by Producing Glyoxylate and Succinate... 

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