Aconitase cofactors

A further way in which metabolic control may be exercised is the artificial deprivation of required ions and cofactors, for example aconitase must have ferrous ions for activity. Conversely, addition of toxic ions is possible, for example aconitase is inhibited by cupric ions. Finally the use of metabolic analogues is possible. If monofluoroacetate is added to cells then monofluorocitrate is produced by titrate synthase and this compound inhibits the activity of aconitase. Great care has to be taken when using metabolic analogues, however, they are often less than 100% specific and may have unexpected and unwanted serious side effects. 

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.

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. 

A brief historical note on the structure of the iron-sulfur clusters in ferredoxins is relevant. After the first analytical results revealed the presence of (nearly) equimolar iron and acid-labile sulfur, it was clear that the metal center in ferredoxins did not resemble any previously characterized cofactor type. The early proposals for the Fe S center structure were based on a linear chain of iron atoms coordinated by bridging cysteines and inorganic sulfur (Blomstrom et al., 1964 Rabino-witz, 1971). While the later crystallographic analyses of HiPIP, PaFd, and model compounds (Herskovitz et al., 1972) demonstrated the cubane-type structure of the 4Fe 4S cluster, the original proposals have turned out to be somewhat prophetic. Linear chains of sulfide-linked irons are observed in 2Fe 2S ferredoxins and in the high-pH form of aconitase. Cysteines linked to several metal atoms are present in metallothionein. The chemistry of iron-sulfur clusters is rich and varied, and undoubtedly many other surprises await in the future. 

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],... 

Fig. 5.3. Protein structure determination by X-ray diffraction. A. Crystals of porcine heart aconitase composed of 754 amino acids. The orthorhombic crystals shown are about 0.5 mm in the longest dimension. B. Film showing the diffraction pattern obtained from the above crystal. These data were used to obtain a 2.7 A resolution structure shown in two representations in panels C and D. Panel C shows the tracing of the protein backbone, with the small molecule (in red and yellow) in the central region depicting the iron-containing cofactor of the enzyme. Panel D shows the space-filling representation. (Courtesy of Dr Arthur H. Robbins, Miles Pharmaceuticals Inc. For details see A.H.Robbins and C.D.Stout (1989). Proteins Structure, Function, and Genetics 5, 289 312.)...

Numerous cellular enzymes and coenzymes require iron, either as an integral part of the molecule or as a cofactor. Notable are the peroxidases and cytochromes, all of which, like Hb, are heme proteins. Other enzymes, such as aconitase and ferredoxin, have iron that is coordinated with sulfur in a so-called iron-sulfiir cluster. Nearly half of the enzymes of the Krehs cycle contain iron. These enzymes and coenzymes, which appear in aU nucleated cells of the body, are referred to collectively as the tissue iron compartment. The tissue iron compartment normally amounts to approximately 8 mg. Although a small compartment, it is metabolicaUy critical. Some iron enzyme activities dimmish early in the course of iron deficiency. ... 

NAD cofactor Citrate synthase Aconitase Citrate production... 

The glycolytic pathway includes three such reactions glucose 6-phosphate isomer-ase (1,2-proton transfer), triose phosphate isomerase (1,2-proton transfer), and eno-lase (yS-elimination/dehydration). The tricarboxylic acid cycle includes four citrate synthase (Claisen condensation), aconitase (j5-elimination/dehydration followed by yS-addition/hydration), succinate dehydrogenase (hydride transfer initiated by a-proton abstraction), and fumarase (j5-elimination/dehydration). Many more reactions are found in diverse catabolic and anabolic pathways. Some enzyme-catalyzed proton abstraction reactions are facilitated by organic cofactors, e.g., pyridoxal phosphate-dependent enzymes such as amino acid racemases and transaminases and flavin cofactor-dependent enzymes such as acyl-C-A dehydrogenases others. 

A wide range of metal ions is present in metalloenzymes as cofactors. Copper zinc snperoxide dismntase is a metalloenzyme that nses copper and zinc to help catalyze the conversion of snperoxide anion to molecnlar oxygen and hydrogen peroxide. Thermolysin is a protease that nses a tightly bonnd zinc ion to activate a water atom, which then attacks a peptide bond. Aconitase is one of the enzymes of the citric acid cycle it contains several iron atoms bonnd in the form of iron-sulfur clusters, which participate directly in the isomerization of citrate to isocitrate. Other metal ions fonnd as cofactors in metalloenzymes include molybdenum (in nitrate rednctase), seleninm (in glutathione peroxidase), nickel (in urease), and vanadinm (in fungal chloroperoxidase). see also Catalysis and Catalysts Coenzymes Denaturation Enzymes Krebs Cycle. 

S-adenosylmethionine (SAM or AdoMet) superfamily, aconitase, and others), enzymes that contain Fe-S heteroatomic clusters (nitrogenase iron-molybdenum cofactor (FeMoco), carbon monoxide dehydrogenase (CODH), and acetyl CoA synthase (ACS)), and enzymes that contain unique ligation sets around specialized iron centers ([NiFe] and [FeFe] hydrogenases) (Figure 1). ... 

Note Another family of serine dehydratases does not use pyridoxal phosphate, but rather an iron-sulfur cluster as the cofactor. These enzymes may use a mechanism similar to the dehydration of citrate catalyzed by aconitase. See Trends Biochem. Sci. 18[ 19931 297-300.)... 

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