Aconitase mechanism

That the aconitase mechanism might involve an electron shift to give an Fe( III Mike iron in the transition state with the substrate bound to it suggests why a whole Fe4S4 cluster is needed. This mechanism would also explain how the substrate triggers the whole mechanism,... 

Gawron, O., Mahajan, K.P. (1966). a-Methyl-cis-aconitic acid, cis-aconitase substrate. II. Substrate properties and aconitase mechanism. Biochemistry 5 2343-50. 

Figure 12. Proposed aconitase mechanism, with structural features deduced from spectroscopic results. (Reproduced with permission from Ref. 130.)...

The mechanism of fluoroacetate toxicity in mammals has been extensively examined and was originally thought to involve simply initial synthesis of fluorocitrate that inhibits aconitase and thereby the functioning of the TCA cycle (Peters 1952). Walsh (1982) has... 

There are several examples in which metabolites that toxify the organism responsible for their synthesis are produced. The classic example is fluoroacetate (Peters 1952), which enters the TCA cycle and is thereby converted into fluorocitrate. This effectively inhibits aconitase—the enzyme involved in the next metabolic step—so that cell metabolism itself is inhibited with the resulting death of the cell. Walsh (1982) has extensively reinvestigated the problan and revealed both the complexity of the mechanism of inhibition and the stereospecihcity of the formation of fluorocitrate from fluoroacetate (p. 239). It should be noted, however, that bacteria able to degrade fluoroacetate to fluoride exist so that some organisms have developed the capability for overcoming this toxicity (Meyer et al. 1990). 

Iron appears to play a significant role in the mechanism of action of aconitase, a constituent enzyme of the tricarboxylic acid cycle (32). 

Another possible pathway of accelerating the in vivo Fenton reaction has been proposed previously [20]. It was suggested that the level of catalytically active ferrous ions may be enhanced as a result of the interaction of superoxide with the [4Fe-4S] clusters of dehydratases such as aconitases. In accord with this mechanism, superoxide reacts with aconitase to oxidize ferrous ion inside of the [4Fe—4S] cluster. In the next step, the remaining ferrous ion is released from the cluster and is capable of participating in Reaction (2) ... 

Gardner and Fridovich [85] proposed that the inactivation of aconitase might be used as an assay of superoxide formation in cells. The mechanism of the interaction of superoxide with aconitases has been considered in Chapter 21. As follows from data presented in that chapter, peroxynitrite is also able to inactivate aconitases rapidly therefore, this method cannot be a specific assay of superoxide detection. 

There have been a number of isolated studies of metal-ion catalyzed nucleophilic reactions of other groupings. Particularly interesting is the induced nucleophilic attack on olefins. Hydration is normally very sluggish. Enzymes can speed up such reactions. Aconitase, an iron-containing enzyme, catalyzes the isomerization of citric acid to isocitric acid, through the intermediacy of cis-aconitic acid. A possible mechanism has been suggested based on the following Co(III) model chemistry. Rapid cyclization of the maleate ester produces Ai and AS chelated malate half ester ... 

Questions concerning the "ferrous-wheel" mechanism came soon after the Villafranca and Mildvan studies with the discovery in Gawron s laboratory that aconitase also contains labile sulfur (23), which, with iron, is indicative of the presence of Fe-S clusters. 

Recently, Sargeson s group has studied the hydration of coordinated carboxy-alkoies as model chemistry for the aconitase reaction (61). A key feature of their nxrdel is the activation of the double bond via carboxylate coordination to the metal and subsequent attack by a coordinated cu-hydroxo group. This was a feature of the "ferrous wheel mechanism as well. The model chemistry suggested that a closer examination of carboxyl labeled substrate and substrate analogues was... 

The details obtained fiom the ENDOR work, in addition to past results, allow for the following mechanism for aconitase. In Scheme II the R group on the substrate is CH2COO, -B represents the amino acid side-chain which stereospeci-fically transfers a proton between citrate and isocitrate, and X is either water or a protein ligand. In the presence of c/s-aconitate, bound water can freely exchange... 

Yeast isopropylmalate isomerase of the leucine biosynthetic pathway, which catalyzes a totally analogous reaction to that of aconitase, converts 3-hydroxy-3-carboxy-4-methylpentanoate to 2-hydroxy-3-carboxy-4-methylpentanoate via an allylic intermediate. In its initial characterization by EPR spectroscopy, a high-field shift in its EPR signal from a g-average of 1.96 to 1.90 is seen upon addition of substrate (70). This result suggests that its mechanism is the same as that found for aconitase. 

The role of 3Fe clusters is open to speculation. Some proteins appear to be functional only as the 3Fe form (e.g., AvFd and fumarate reductase). For others the 3Fe form may be an unfortunate side effect of having a reactive readily displaced ligand which is required for the enzyme mechanism as in aconitase. Because the 3Fe cluster can take up other metals (e.g., nickel, cadmium, or zinc) (Moura et al., 1986 Surerus et ai, 1987 Surerus, 1989), it is possible that the 3Fe form may be the precursor for... 

NO also has cytotoxic effects when synthesized in large quantities, eg, by activated macrophages. For example, NO inhibits metalloproteins involved in cellular respiration, such as the citric acid cycle enzyme aconitase and the electron transport chain protein cytochrome oxidase. Inhibition of the heme-containing cytochrome P450 enzymes by NO is a major pathogenic mechanism in inflammatory liver disease. 

In addition to aconitases, nitric oxide is an inhibitor of many other enzymes such as ribonucleotide reductase [71], glutathione peroxidase [72,73], cytochrome c oxidase [74], NADPH oxidase [75], xanthine oxidase [76], and lipoxygenase [77] but not prostaglandin synthase [78]. (Mechanism of lipoxygenase inhibition by nitric oxide is considered in Chapter... 

If this mechanism is correct, the aconitase reaction is an excellent illustration of the influence of the stereochemistry of the metal, as well as its charge, upon the course of a biochemical reaction. The charge on the iron is, of course, responsible for the formation of the resonating carbonium ions A and B from C, D, or E. In C and D the flow of electrons toward iron severs the bond between carbon and the hydroxyl group, whereas in E the proton is released from coordinated water and attached to one of the two ethylenic carbon atoms. The stereochemistry of the iron atom can be credited with holding the organic molecule and the hydroxide in their proper spatial relationship in A and B. It has been recently demonstrated that the complexes of the aconitase substrates with nickel have the structures postulated by Speyer and Dickman and shown in Figure 3 (19). 

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... 

Fig. 4 Mechanism of aconitase.4 The aci-carboxylate intermediate shares a low-barrier hydrogen bond with the Fe-OH group.

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