Aconitase scheme

By this time it was demonstrated that the [3Fe-4S]W+ form of aconitase is inactive, while the [4Fe-4S]2+ form is active. How is the activity of the enzyme affected by the oxidation state of the [4Fe-4S] cluster Because the active enzyme contains a [4Fe-4S]2+ cluster, either the 3+ or 1+ oxidation states may also be stable. The 3+ state is unstable since oxidation of the [4Fe-4S]2+ resulted in the immediate loss of a ferrous ion and conversion to a [3Fe-4S]i+ cluster (46,47). However, reduction of active aconitase by sodium dithionite or photoreduction in the presence of deazaflavin produced in high yields an EPR signal characteristic for [4Fe-4S]l+ clusters (47). When active enzyme within an anaerobic assay cuvette was photoreduced, the activity of the enzyme dropped to 1/3 of its initial value. Further photoreduction resulted in cluster destruction. Then, if the enzyme is reoxidized with air, the activity returned to its original value. This demonstrated that the redox state of the cluster can modulate the enzyme activity. A scheme summarizing the cluster interconversions and various redox states of the Fe-S cluster of aconitase is shown below. 

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... 

In contrast, selective inhibition of enzyme activity involves highly specific interactions between the protein and chemical groups on the xenobiotic. An excellent example of this type of inhibition is seen in the toxic effect of fluoroacetate, which is used as a rodenticide. Although fluoroacetate is not directly toxic, it is metabolized to fluoroacetyl-CoA, which enters the citric acid cycle due to its structural similarity to acetyl-CoA (Scheme 3.5). Within the cycle, fluoroacetyl-CoA combines with oxalo-acetate to form fluorocitrate, which inhibits the next enzyme, aconitase, in the cycle [42]. The enzyme is unable to catalyze the dehydration to cis-aconitate, as a consequence of the stronger C-F bond compared with the C-H bond. Therefore, fluorocitrate acts as a pseudosubstrate, which blocks the citric acid cycle and, subsequently, impairs ATP synthesis. 

Scheme 3.5 Metabolism of fluoroacetate to fluorocitrate, a specific inhibitor of aconitase. Modified from [42].

The enzyme aconitase catalyzes the isomerization of citric acid to isocitric acid via the intermediate cis-aconitic acid (Scheme 46),530 and various attempts have been made to model this reaction.21 The cobalt Ill) complexes derived from methyl maleate (171) and methyl fumarate (172) have been prepared531 to study intramolecular attack by coordinated hydroxide on the alkene. Generation of the hydroxo species of the maleic acid complex leads to rapid cyclization to give the... 

Figure 3 The catalytic cycle of aconitase. The circle symbolizes the [3Feb-4S] structure that carries the reactive Fea. Note that the right-hand side of the scheme is the mirror image of the left-hand side with respect to the CaCpCT fragment.

If the methyl carbon atom of pyruvate is labeled with which of the carbon atoms of oxaloacetate would be labeled after one turn of the citric acid cycle (See the lettering scheme for oxaloacetate in Figure 17.1 in this book.) Note that the new acetate carbons are the two shown at the bottom of the first few structures in the cycle, because aconitase reacts stereospecifically. 

Scheme 6 Some of the important iron-sulfur cluster units found in metalloenzymes. [2Fe-2S] rhombus cluster is characteristic of [2Fe-2S] feiredoxins and Rieskie proteins, [4Fe-4S] cubane -e.g., in [4Fe-4S] feiredoxins, aconitase [3Fe-4S] clusters are present in the inactive form of aconitase, [3Fe-4S] feiredoxins. The iron vertices, designated as [Fe], have high-spin tetrahedral FeS4 coordination. Reprinted from [140], with the permission of Elsevier, copyright 2000...

The enzyme (aconitase), being itself chiral, can distinguish between the two enantiotopic carboxymethylene groups (one pro-R and the other pro-S) of citrate (Figure 8.20). Then, as shown in Scheme 8.86, the pro-R proton from the pro-R carboxymethylene is abstracted and the hydroxyl is lost in an antiperiplanar... 

Scheme 8.86. A cartoon representation of the eqnilibrium between citrate and isocitrate, occurring through cw-aconitate and as catalyzed by the enzyme aconitase (see Glusker, J. P. Aconitase, in Boyer, P. D. (ed.), Enzymes,Wo. 5,3rd edition. Academic Press, New York, 1971, 413).

Then, continuing in Scheme 11.89, isomerization of citrate (aconitase, aconitate hydratase, EC 4.2.1.3) to isocitrate occurs. This isomerization passes through... 

---