Citrate cis-aconitate

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). 

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... 

Gawron et al. (13,14) determined the stereochemistry of natural isocitric acid by chemical means. The results require the rrons-addition of water across the cis-aconitate intermediate double bond to produce either citrate or 2R,3S-isodtrate. Mass and NMR analyses of isotopically labeled citrate and isocitrate in the early 1960 s (15-17), defined the stercospedficities of the dehydration steps. These results led Gawron to propose the binding of cis-aconitate to the active site in two orientations differing by a 180° rotation about the double bond, as shown in Equation 2. This allows for the protonation by a base (-BH) and hydroxylation of the double bond to occur on aconitase at single, separate loci for the formation of either citrate or isocitrate. 

Citrate is subsequently isomerized to isocitrate this involves dehydration and rehydration via the intermediate cis-aconitate. Both reactions are... 

Significant inhibition of all activities below pH 7 by citrate for which a Ki value of 5.2 mil/ has been observed at pH 6.5. Isocitrate much less effective, and only minor effects noted with cis-aconitate, f-malate, succinate, ar ketoglutarate, and oxalate. Malonate, tartrate, L-glutamate, glutarate, adipate, 0-hydroxy-0-methyl glutaryl-CoA, and 0-hydroxybutyrate also found ineffective as instantaneous inhibitors... 

Fig. 5.22. Oxidation of acetyl-CoA via the tricarboxylic acid (TCA) cycle. Individual enzymes of the pathway are marked. 1, citrate synthase 2 and 3, cis-aconitate hydratase 4 and 3, isocitrate dehydrogenase 6, a-oxo glutarate dehydrogenas 7, succinate thiokinase 8, succinate...

Bromate, chloride, bromide, nitrite, nitrate, hypophosphite (HP022 ), selenite, selenate, sulphate, phosphate, pyrophosphate, arsenate, chromate, a-hydroxybutyrate, butyrate, formate, acetate, glycolate, gluconate, valerate, a-hydroxy valerate, pyruvate, monochloroacetate, dichloroacetate, trifluoroacetate, galactonurate, gluconurate, a-keto-glutarate, oxalate, fumarate, phthalate, oxalacetate, citrate, isocitrate, cis aconitate, trans aconitate, succinate, maleate, malonate, quinate, tartrate, hexane sulphonate, octane sulphonate, octane sulphate, decane sulphonate, dodecane sulphonate and dodecane sulphate... 

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. 

Step 2 involves the dehydration of citrate to cir-aconitate followed by the hydration of cis-aconitate to isocitrate. Aconitase catalyzes these reversible reactions. 

Transfer of citrate through the inner membrane of MCh is provided by a tricarboxylate transporter (m.w. 32.5 kD), which also catalyzes transport of treo-Ds-isocitrate, cis-aconitate and other tricarboxylates (LaNoue and School-werth, 1979 Kaplan et al, 1990). This is electroneutral exchange for either another tricarboxylate or dicarboxylate (e.g. malate or succinate), or for phosphoenolpyruvate. Formation of glutathione-citryl thioester is irreversibly inhibited by (-)erythrofluorocitrate (IC50 = 25 pmol FC/mg protein), which makes a stable adduct with the synthase (Kun et al, 1977). However, the block of citrate transport... 

The tertiary hydroxyl group is not properly located in the citrate molecule for the oxidative decarboxylations that follow. Thus, citrate is isomerized into isocitrate to enable the six-carbon unit to undergo oxidative decarboxylation. The isomerization of citrate is accomplished by a dehydration step followed by a hydration step. The result is an interchange of a hydrogen atom and a hydroxyl group. The enzyme catalyzing both steps is called aconitase because cis-aconitate is an intermediate. 

Although citrate has been excluded as the primary condensation product of pyruvate and oxalacetate, no direct evidence bearing upon the nature of this product has as yet been obtained. The participation of cfs-aconitic and isocitric acids is speculative. Nor is there any evidence supporting the hypothesis that pyruvate and oxalacetate condense to form a hypothetical intermediate oxalcitraconic acid which can be oxidatively decarboxylated to citric acid. Since citrate, aconitate and isocitrate are in equilibrium with each other, the participation of the last two substances as intermediates of carbohydrate oxidation would, on the surface, appear to be doubtful. Krebs, however, believes that the conversion of cis-aconitate to a-ketoglutarate occurs so rapidly in liver that equilibrium with citrate is not attained. 

Unfortunately the resulting citrate is a tertiary alcohol which cannot be readily oxidized. Aconitase catalyzes the rearrangement of citrate to give an oxidizable secondary alcohol. This reaction involves an elimination/addition sequence, catalyzed by an iron-sulphur cluster (Fe4S4), with an alkene intermediate, cis-Aconitate ... 

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... 

It is known that the enzyme aconitase catalyzes (1) the dehydration of both citrate and isocitrate to form cis-aconitate, (2) the reverse reactions, and (3) the interconversion of citrate and isocitrate thusly ... 

Other C-0 lyase enzymes include aconitate hydratase or aconitase (E. C. 4.2.1.3), an enzyme that catalyzes two tricarboxylic acid cycle steps from isocitric acid to citrate (14)1141 or vice versa, via the intermediate cis-aconitate (13). Citrate dehydratase (E. C. 4.2.1.4) is only capable of converting citrate to cis-aconitate and does not act on isocitrate (15) 115l... 

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