Aconitase active site structure

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...

Figure 5 Structures of the (a) Fe3S4(Cys)4 ferredoxin site (PDB 7FD1) (b) aconitase active site with bound trans-aconitate (PDB lACO) (c) NiFe4Ss cluster in carbon monoxide dehydrogenase (PDB IJQK).

One might also wish to compare the structures and conformations of substrates and inhibitors of an enzyme. Citrate and isocitrate are two ions with slightly different formulae and different conformations. As they are both substrates of the same enzyme (the Krebs cycle enzyme aconitase), a comparison between them would be expected to lead to information on how each is bound in the active site of the enzyme. This comparison, shown in Figure 16.2, provided some insight into the reasons... 

Aconitase was initially crystallized in the [3Fe-4S]" " form, and these crystals could be converted to the [4Fe S] " " form by addition of ferrous ammonium sulfate. Alternatively, anaerobic crystallization methods were used to crystallize the [4Fe-4S] " " form directly." The iron-sulfur cluster of aconitase sits within a solvent-filled cleft in the protein structure and is coordinated by Cys 358, Cys 421, and Cys 424. The [3Fe S]+ cluster shows the typically cuboidal geometry, with the open iron site directed toward the active site cleft. Conversion to the active [4Fe-4S] " " form results in almost no structural perturbation, with the extra iron atom simply being inserted into the vacant site in the cluster. The fourth iron is tetrahedrally coordinated, but with a solvent hydroxide rather than a cysteine as the fourth ligand. (The electron density clearly reveals a bound solvent, and previous ENDOR studies had demonstrated that the bound species was associated with only a single proton.)... 

Once substrate is bound, a base in the active site, thought to be the alkoxide form of serine 642," abstracts a proton from either Ca (in the case of citrate) or C/3 (in the case of isocitrate). This proton abstraction produces initially a carbanion intermediate, depicted here as the uci-acid. The fact that the nitro analogs of the substrates are both good structural mimics of the putative <2ci-acid intermediates and are also good inhibitors of aconitase provides additional evidence for the intermediacy of the uci-acid intermediates. The collapse of the uci-acid intermediate ultimately... 

Diagrammatic representation of prochirality of citrate. (A) Structure of citrate, (B) Three point landing of citrate on the active site of aconitase. Only one of the CHfiOO- groups, the one which is invohmd in binding, is attacked by aconitase. This is why only one form of labeled a-ketoglutarate is produced. [(Ref. Ogston, AG., Nature, 162 963(1948) ... 

It is now widely appreciated that the old, established view of Fe-S clusters as a class of integral structural units whose activity is limited to electron transfer is in error. We have become aware of other roles. For example, in the Fe-hydrogenases, it is certain that a cluster serves as the catalytic site with which the Hj/H substrates interact [164]. Moreover, following the discovery by Beinert s group that aconitase is an Fe-S protein [175], there are now further examples of clusters occurring in enzymes that do not catalyze, at least obviously, a redox reaction. With aconitase itself, it is established that substrates bind to one Fe subsite of a [4Fe-4S] cluster without major disturbance of the core structure [189]. Such findings demonstrate that real chemistry is available, reactivity that may be exploited once tuned by the protein environment. 

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