Aconitase, function

T. A. Rouault, C.D. Stout, S. Kaptain, J.B. Harford, and R.D. Klausner. 1991. Structural relationship between an iron-regulated RNA-binding protein (IRE-BP) and aconitase Functional implications Cell 64 881-883. (PubMed)... 

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... 

To successfully describe the structure and function of nitrogenase, it is important to understand the behavior of the metal-sulfur clusters that are a vital part of this complex enzyme. Metal-sulfur clusters are many, varied, and usually involved in redox processes carried out by the protein in which they constitute prosthetic centers. They may be characterized by the number of iron ions in the prosthetic center that is, rubredoxin (Rd) contains one Fe ion, ferredoxins (Fd) contain two or four Fe ions, and aconitase contains three Fe ions.7 In reference 18, Lippard and Berg present a more detailed description of iron-sulfur clusters only the [Fe4S4] cluster typical of that found in nitrogenase s Fe-protein is discussed in some detail here. The P-cluster and M center of MoFe-protein, which are more complex metal-sulfur complexes, are discussed in Sections 6.5.2. and 6.5.3. 

Proteins containing iron-sulfur clusters are ubiquitous in nature, due primarily to their involvement in biological electron transfer reactions. In addition to functioning as simple reagents for electron transfer, protein-bound iron-sulfur clusters also function in catalysis of numerous redox reactions (e.g., H2 oxidation, N2 reduction) and, in some cases, of reactions that involve the addition or elimination of water to or from specific substrates (e.g., aconitase in the tricarboxylic acid cycle) (1). 

Since this reaction requires no oxidation-reduction chemistry, the finding 15 years ago that aconitase is an Fe-S protein was quite unexpected. Until recently a paradigm for Fe-S proteins has been that they function primarily in reactions requiring electron transfer. Accordingly, the central question in the study of aconitase for the past several years has been What is the function of its Fe-S cluster ... 

Why did nature use an Fe-S cluster to catalyze this reaction, when an enzyme such as fumarase can catalyze the same type of chemistry in the absence of any metals or other cofactors One speculation would be that since aconitase must catalyze both hydrations and dehydrations, and bind substrate in two orientations, Fe in the comer of a cubane cluster may provide the proper coordination geometry and electronics to do all of these reactions. Another possibility is that the cluster interconversion is utilized in vivo to regulate enzyme activity, and thus, help control cellular levels of citrate. A third, but less likely, explanation is that during evolution an ancestral Fe-S protein, whose primary function was electron transfer, gained the ability to catalyze the aconitase reaction through random mutation. 

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. 

DEGREE OF DISSOCIATION HENDERSON-HASSELBALCH EQUATION ACID-BASE EQUILIBRIUM CONSTANTS BR0NSTED THEORY LEWIS ACID ACIDITY FUNCTION LEVELING EFFECT ACIDITY FUNCTION ACID-LABILE SULFIDES ACID PHOSPHATASE ACONITASE... 

A representative sampling of non-heme iron proteins is presented in Fig. 3. Evident from this atlas is the diversity of structural folds exhibited by non-heme iron proteins it may be safely concluded that there is no unique structural motif associated with non-heme iron proteins in general, or even for specific types of non-heme iron centers. Protein folds may be generally classified into several categories (i.e., all a, parallel a/)3, or antiparallel /8) on the basis of the types and interactions of secondary structures (a helix and sheet) present (Richardson, 1981). Non-heme iron proteins are found in all three classes (all a myohemerythrin, ribonucleotide reductase, and photosynthetic reaction center parallel a/)8 iron superoxide dismutase, lactoferrin, and aconitase antiparallel )3 protocatechuate dioxygenase, rubredoxins, and ferredoxins). This structural diversity is another reflection of the wide variety of functional roles exhibited by non-heme iron centers. 

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... 

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). 

It is now clear that in addition to their widespread involvement in electron transfer pathways, iron-sulfur clusters function as catalytic centers in a wide variety of enzymes. The first example of such an enzyme is aconitase. It was at first thought that the role of the iron-sulfur group was regulatory, but it is now clear that in this enzyme the iron-sulfur group is part of the catalytic site. One of the iron atoms can coordinate water or hydroxyl and plays a key role in the isomerization catalyzed by the enzyme (Emptage et al., 1983). 

Nitric oxide may be the active moiety of STZ that induces diabetes in this animal model. STZ contains a nitroso moiety and may release nitric oxide by a process analogous to the nitric oxide donor compounds SIN-1 and nitroprusside. Turk et al. (1993) have shown that incubation of rat islets with STZ at concentrations that impair insulin secretion results in the generation of nitrite and the accumulation of cGMP. STZ also inhibits mitochondrial aconitase activity of islets to a degree similar to that achieved by IL-1. These findings provide the first evidence that STZ impairs islet function by liberating nittic oxide. 

Our studies with mitochondrial respiration also ruled out the hypothesis that NO-induced impairment of mitochondrial function accounted for the decrease in protein synthesis. The reasons for this conclusion include first, a minimal decrease in aconitase activity and essentially no decrease in complex 1 or complex II activity was seen in HC exposed to KC supernatant or cytokines -I- LPS, despite a marked reduction in protein synthesis and second, exposure of HC to NO resulted in a profound and prolonged decrease in HC protein synthesis (up to 18 hr), but a very short-lived decrease in mitochondrial respiration (90 min)... 

Rodent KC and HC, as well as human HC, express an inducible NO synthase under septic or inflammatory conditions. In vivo in endotoxemia, this expression is transient. Our in vivo data indicate that this induced -NO serves a protective role in the liver and reduces hepatic injury in endotoxemia. This protective action may be mediated by the capacity of NO to neutralize oxygen radicals and prevent platelet adherence and aggregation. Our in vitro studies show that HC-derived -NO can activate soluble guanylate cyclase. Other in vitro effects include the nonspecific suppression of protein synthesis and a small reduction in mitochondrial aconitase activity. The relevance of these in vitro actions to hepatic function in vivo remains to be determined. 

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