Krebs Aconitase

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. 

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 Enzyme Aconitase. The enzyme aconitase catalyzes the elimination or addition of water in the second step of the citric acid (Krebs) cycle, catalyzing the interconversion of citrate and isocitrate via cix-aconitate. See reference 8, pages 190-196, Figure 7.49, and equation 7.13. 

Figure 9.2 Summary of reactions of the Krebs cycle. The names of the enzymes are dtrate synthase, aconitase, isodtrate dehydrogenase (there are two enzymes, one ubTizes NAD as the cofactor, the other NADPT it is assumed that the NAD -specific enzyme is that involved in the cycle), oxoglutarate dehydrogenase, sucdnyl CoA synthetase, succinate dehydrogenase, fumarate hydratase, malate dehydrogenase.

Citric acid has three prochiral centres The Krebs cycle is a process involved in the metabolic degradation of carbohydrate (see Section 15.3). It is also called the ciU ic acid cycle, because citric acid was one of the first intermediates identified. Once formed, citric acid is modified by the enzyme aconitase through the intermediate... 

Krebs cycle Inhibition of aconitase by superoxide and fluoroacetate, of succinate dehydrogenase by methamphetamine and mal-onate, of alpha-ketoglutarate dehydrogenase by salicylic add... 

As shown in Table V, a number of Fe S-containing proteins perform reactions other than redox or electron transfer. That is, the function of the cluster does not include a change in oxidation state, even as a transient step in catalysis. This role is best illustrated by aconitase, one of the most extensively studied Fe S proteins, regardless of function. The elegant recent work on this enzyme is largely under the guiding hand of H. Beinert and is summarized in the Krebs Memorial Lecture (Beinert and Kennedy, 1989). 

The toxicity of fluoroacetic acid and of its derivatives has played an historical decisive role at the conceptual level. Indeed, it demonstrates that a fluorinated analogue of a natural substrate could have an activity profile that is far different from that of the nonfluorinated parent compound. The toxicity of fluoroacetic acid is due to its ability to block the citric acid cycle (Krebs cycle), which is an essential process of the respiratory chain. The fluoroacetate is transformed in vivo into 2-fluorocitrate by the citrate synthase. It is generally admitted that aconitase (the enzyme that performs the following step of the Krebs cycle) is inhibited by 2-fluorocitrate the formation of aconitate through elimination of the water molecule is a priori impossible from this substrate analogue (Figure 7.1). 

Welsh, N., Eizirik, D. L., Bendtzen, K., and Sandler, S. (1991a). Interleukin-1/3-induced nitric oxide production in isolated rat pancreatic islets requires gene transcription and may lead to inhibition of Krebs cycle enzyme aconitase. Endocrinology (Baltimore) 129, 3167-3173. 

This naturally occurring toxicant is an analogue of acetate and is incorporated into acetyl CoA (fluoroacetate) and hence into Krebs cycle (TCA cycle) as fluorocitrate. This blocks the enzyme aconitase, as the fluorine atom cannot be removed. The TCA cycle is blocked, and citrate accumulates. The mitochondrial energy supply is disrupted, hence cardiac damage occurs. Lack of oxaloacetate will allow ammonia to accumulate leading to convulsions. 

Fig. 3. Krebs citric acid cycle. Enzymes involved (1) Condensing enzyme (2) aconitase (3) isocitric acid (4) a-ketoglucaric acid dehydrogenase (4) a succinic acid thiokinasc (5) succinic acid dehydrogenase (6) fumarasc (7) malaic acid dehydrogenase. Abbreviations CA = citric acid ACOM = eij-aconitic acid KG = a-ketoglutaric acid SIC = succinic acid FA = fumaric acid MA = malic acid OA = oxalaceiic acid...

Krebs cycle intermediates (Table 5.12) and/or enzymes (Table 5.13). Nevertheless, certain key enzymes, especially aconitase and isocitrate dehydrogenase, are very low in activity or are undetectable in species such as H. diminuta, whereas only very small amounts of 14C02, a characteristic end-product of the TCA cycle, were liberated in vitro from [14C]glucose by the tetrathyridia of Mesocestoides corti (399) and adults of Cotugnia digonopora (618). The classical TCA cycle is, therefore, unlikely to function to any significant extent in these cestodes. 

There are two Krebs cycle inhibitors that are worth mentioning. Malonate inhibits succinate dehydrogenase because of its very similar structure. Fluoro-acetate inhibits cis-aconitase, which is an Fe-S enzyme. The fluoroacetate replaces acetate as a substrate in the citrate synthase reaction when this combines with cis-aconitase, however, no further reaction becomes possible. 

Aconitase Iron-Sulfur Mitochondria Isomerase in Krebs cycle... 

One might also wish to compare the structures and conformations of substrates and inhibitors of an enzyme. Citrate and isocitrate are two ions with slightly different formulae and different conformations. As they are both substrates of the same enzyme (the Krebs cycle enzyme aconitase), a comparison between them would be expected to lead to information on how each is bound in the active site of the enzyme. This comparison, shown in Figure 16.2, provided some insight into the reasons... 

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-... 

Iron response elements consist of short stretches of ribonucleic acid, and they occur in the mRNA coding for ferritin, transferrin receptor, and 8-aminolevulinic acid s)mthase. Each IRE can be bound by an IRP. Several different IRPs exist. Surprisingly, one of the IREs is quite similar to aconitase, the Krebs cycle enzyme (Kim et ah, 1996 Toth and Bridges, 1995 Henderson et ah, 1996). All IRPs contain an iron binding site. This site consists of several residues of cysteine. To be more specific, the sulfhydryl groups of cysteine residues function to bind iron atoms. 

Biotransformation, especially phase I metabolic reactions, cannot be assumed to be synonymous with detoxification because some drugs (although a minority) and xenobiotics are converted to potentially toxic metabolites (e.g. parathion, fluorine-containing volatile anaesthetics) or chemically reactive intermediates that produce toxicity (e.g. paracetamol in cats). The term lethal synthesis refers to the biochemical process whereby a non-toxic substance is metabolically converted to a toxic form. The poisonous plant Dichapetalum cymosum contains monofluoroacetate which, following gastrointestinal absorption, enters the tricarboxylic acid (Krebs) cycle in which it becomes converted to monofluorocitrate. The latter compound causes toxicity in animals due to irreversible inhibition of the enzyme aconitase. The selective toxicity of flucytosine for susceptible yeasts (Cryptococcus neoformans, Candida spp.) is attributable to its conversion (deamination) to 5-fluorouracil, which is incorporated into messenger RNA. 

Substances of toxicological significance Monofluoro-acetate Aconitase (tricarboxylic acid, (,Dichapetalum Krebs cycle) cymosum) No clinical uses Becomes incorporated into fluoroacetyl coenzyme A, which condenses with oxaloacetate to form fluorocitrate ( lethal synthesis )... 

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... 

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