Aconitase hydratase

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. 

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.

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. 

Aconitate hydratase [Fe4S4]— aconitase Phosphopyruvate hyd ratase— enolase ... 

The citrate ion, a very important prochiral metabolic intermediate, has three prochiral centers at C-2, C-3, and C-4, respectively. That at C-3 distinguishes the pro-R and pro-S arms and determines the stereochemical numbering. Citrate containing 14C in the sn-1 position is called s -citrate[l-14C] and is the form of labeled citrate that is synthesized in living cells from oxaloacetate and [l-14C]acetyl coenzyme A (see Fig. 10-6). The first step in the further metabolism of citrate is the elimination of the -OH group from C-3 together with the Hr proton from C-4 through the action of the enzyme aconitate hydratase (aconitase). In this case the proton at C-4 (in the pro-R arm) is selected rather than that at C-2. 

Two consecutive reactions of the citric acid cycle (Fig. 10-6), the dehydration of citrate to form czs-aconi-tate and the rehydration in a different way to form isocitrate (Eq. 13-17), are catalyzed by aconitase (aconi-tate hydratase). Both reactions are completely stereospecific. In the first (Eq. 13-17, step a), the pro-R proton from C-4 (stereochemical numbering) of citrate is removed and in step c isocitrate is formed. Proton addition is to the re face in both cases. 

Other enzymes in the aconitase family include isopropylmalate isomerase and homoaconitase enzymes functioning in the chain elongation pathways to leucine and lysine, both of which are pictured in Fig. 17-18.90 There are also iron-sulfur dehydratases, some of which may function by a mechanism similar to that of aconitase. Among these are the two fumarate hydratases, fumarases A and B, which are formed in place of fumarase C by cells of E. coli growing anaerobically.9192 Also related may be bacterial L-serine and L-threonine dehydratases. These function without the coenzyme pyridoxal phosphate (Chapter 14) but contain iron-sulfur centers.93-95 A lactyl-CoA... 

Among the most deadly of simple compounds is sodium fluoroacetate. The LD50 (the dose lethal for 50% of animals receiving it) is only 0.2 mg/kg for rats, over tenfold less than that of the nerve poison diisopropylphosphofluoridate (Chapter 12).a b Popular, but controversial, as the rodent poison "1080," fluoroacetate is also found in the leaves of several poisonous plants in Africa, Australia, and South America. Surprisingly, difluoroacetate HCF2-COO is nontoxic and biochemical studies reveal that monofluoroacetate has no toxic effect on cells until it is converted metabolically in a "lethal synthesis" to 2R,3R-2-fluorocitrate, which is a competitive inhibitor of aconitase (aconitate hydratase, Eq. 13-17).b This fact was difficult to understand since citrate formed by the reaction of fluorooxalo-acetate and acetyl-CoA has only weak inhibitory activity toward the same enzyme. Yet, it is the fluorocitrate formed from fluorooxaloacetate that contains a fluorine atom at a site that is attacked by aconitase in the citric acid cycle. 

Hydration and/or dehydration reactions are frequently catalyzed by metallopro-teins. Examples are proteins containing nickel (urease), zinc (e.g., peptidases), molybdenum (the hydratase partial reaction of formate oxidoreductase), tungsten (acetylene hydratase). An obvious difference between Ni, Zn, on the one hand, and Fe, Mo, W, on the other, is that the first are directly coordinated to the protein whereas the latter are also part of a cofactor. With reference to the Fe/S cluster in aconitase it has been suggested that cofactor coordination may provide an added flexibility to the active site, in particular to the substrate binding domain [15],... 

Aconitase is the trivial name for citrate dehydratase cw-aconitate hydratase (EC 4.2.1.3). It catalyzes the reversible isomerization reaction of citrate into isocitrate via the intermediate cA-aconitate (Figure 2). It is a water-soluble, monomeric protein. In eukaryotic cells aconitase is located in the mitochondrial matrix. In prokaryotes the enzyme occurs in the cytoplasma. The pig heart enzyme consists of 754 amino-acid residues, providing a molecular mass of 83 kDa [27], Aconitase from other sources has similar size. The porcine protein is synthesized with a mitochondrial targeting sequence. The mature, functional protein can be (over)expressed in Escherichia coli [28],... 

These are the most widely encountered iron proteins. They display important biological roles as redox centers (ferredoxins, rubredoxins)93 and chemical catalysts94 (aconitase,95 several (de)hydratases, sulfite reductase,96 hydrogenase,97 CO dehydrogenase,98 and nitrogenase). 

Aconitase has been a particularly fertile protein for investigating and establishing cluster transformations. Aerobic isolation of aconitase or air-exposure of the active [4Fe-4S] + + form results in an inactive enzyme containing a [3Fe-4S]+ cluster. However, under reducing conditions the [3Fe-4S]° cluster avidly takes up Fe to reform the catalytically active [4Fe-4S] + + cluster. The [4Fe-4S] clusters in (de)hydratases such as aconitase and ferredoxins which have noncysteinyl ligation at a specific Fe atom are particularly susceptible to this type of reversible cluster... 

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... 

Formation of IsocItrate via cIs-Aconltate The enzyme aconitase (more formally, aconitate hydratase)... 

Figure 2 CrystallographicaUy defined active-site structures for Fe-S centers involved with substrate binding and activation. Stmctures are taken from coordinates deposited in the Protein Data Bank (a) Fe NHase, PDB ID 2AHJ, NO-bound form of Rhodococcus erythropolisnitnie hydratase (b) [4Fe-4S] + isocitrate, PDB ID 7ACN, isocitrate-bound form of porcine heart aconitase (c) Fe SOR, PDB ID IDQK, reduced Pyrococcus furiosvs (d) [4Fe-4S] + SAM, PDB ID lOLT, SAM-bound Escherichia co//HemN (e) Ni-Fe H2ase, PDB...

---