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=Aconitase= | =Aconitase= | ||
Aconitase (PDB [[7acn]]) | Aconitase (PDB [[7acn]]) is a single polypeptide (M<sub>r</sub> 83kD) that catalyzes the reversible isomerization of citrate and isocitrate.<ref name="Zheng">Zheng, L., Kennedy, MC., Beinert, H., Zalkin, H. "Mutational analysis of active site residues in pig heart aconitase." J Biol Chem 1992, 267, 7895-7903.</ref> It is the second enzyme in the Citric acid cycle, which is a series of enzyme-catalysed chemical reactions that is crucial to aerobic cellular respiration and the production of ATP. | ||
==Structure== | |||
The <scene name='Anthony_Noles_Sandbox/Secondary_structure/1'>secondary structure</scene> consists of numerous alternating alpha helices and beta sheets (SCOP classification α/β alternating). The tertiary structure is somewhat bilobed with the active site in the middle, and, since there is only one subunit, there is no quaternary structure. Aconitase consists of four domains, three of which are tightly packed while the fourth is more flexible. <ref name="Frishman">Frishman, D., and Hentze, M.W., "Conservation of aconitase residues revealed by multiple sequence analysis: Implications for structure/function relationships." European Journal of Biochemistry, 1996, 239, 197-200.</ref> Aconitase contains a <scene name='Anthony_Noles_Sandbox/Fe-scluster/2'>4Fe-4S iron-sulfur cluster</scene>. This iron sulfur cluster does not participate in redox as most do, but holds the OH group of citrate to facilitate its elimination.<ref>PMID:16407072 </ref> It is at this 4Fe-4S site that catalysis occurs and citrate or <scene name='Anthony_Noles_Sandbox/Fe-scluster_bound_isocitrate/8'>isocitrate</scene> is bound. The rest of the <scene name='Anthony_Noles_Sandbox/Fe-scluster_w_active_site/5'>active site (manually rotate this scene to see the proximity of each residue to the 4Fe-4S cluster)</scene> is made up of residues Gln72, Asp100, His101, Asp165, Ser166, His167, His147, Glu262, Asn258, Cys358, Cys421, Cys424, Cys358, Cys421, Asn446, Arg447, Arg452, Asp568, Ser642, Ser643, Arg644, Arg580. <ref name="Beinert">Beinert, H., Kennedy, M. C., Stout, C.D. “Aconitase as Iron−Sulfur Protein, Enzyme, and Iron-Regulatory Protein.” Chem. Rev. 1996, 96, 2335−2373.</ref> | |||
{{STRUCTURE_7acn | PDB=7acn | SCENE= }} | |||
==Mechanism of Aconitase== | ==Mechanism of Aconitase== | ||
Substrate-free aconitase contains a [4Fe-4S]<sup>2+</sup> cluster with hydroxyl bound to one of the Fe. Upon binding of substrate the bound hydroxyl is protonated. A hydrogen bond from <scene name='Anthony_Noles_Sandbox/His101/3'>His101</scene> to the isocitrate hydroxyl is donated to form water. Alternatively, the proton could be donated by <scene name='Anthony_Noles_Sandbox/His167/3'>His167</scene> as this histidine is hydrogen bonded to a H<sub>2</sub>O molecule. His167 is also hydrogen bonded to the bound H<sub>2</sub>O in the [4Fe-4S] cluster. Both <scene name='Anthony_Noles_Sandbox/His_101_and_167/4'>His101 and His167</scene> are paired with carboxylates (<scene name='Anthony_Noles_Sandbox/Asp100_and_glu262/3'>Asp100 and Glu262</scene>, respectively) and are likely to be protonated. The conformational change associated with substrate binding reorients the cluster. <ref name="Beinert" /> The residue which removes a proton from citrate or isocitrate is <scene name='Anthony_Noles_Sandbox/Ser642/4'>Ser642</scene>. <ref name="Beinert" /> This causes the cis-Aconitate intermediate (seen below), which consists of a double bond, which is a direct result of the deprotonation. Then, there is a rehydration of the double bond of cis-aconitate to form isocitrate (if the original substrate was citrate). To better understand this, consider this process as stages, seen below. | |||
Substrate-free aconitase contains a [4Fe-4S]2+ cluster with hydroxyl bound to one of the Fe. Upon binding of substrate the bound hydroxyl is protonated. A hydrogen bond from <scene name='Anthony_Noles_Sandbox/His101/ | |||
====Stage 1: Dehydration==== | ====Stage 1: Dehydration==== | ||
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[[ Image:Aconitase.JPG]] | [[ Image:Aconitase.JPG]] | ||
== | ==Regulation== | ||
Aconitase can be inhibited or activated to increase or decrease the ability to catalyze the reaction of citrate to isocitrate. The activity of aconitase can be reduced when one Fe is lost from the cluster. This lowers the activity over 100-fold, but then can regain full activity by adding another Fe from solution. <ref name="Flint">Flint, DH., and Allen, RM. "Iron-sulfur protein with nonredox functions.” Chem. Rev. 1996, 96, 2315−2334.</ref> Aconitase is also strongly inhibited by nitro analogs <ref name="Flint" /> | |||
The Citric Acid Cycle works in such a way that the product of one reaction becomes the reactant of another, with different enzymes catalyzing each reaction. Aconitase is one such enzyme. Some of these enzymes are tightly regulated, either activated or inhibited, by the concentration of reactant, product, ATP or NADH, and thus are rate-determining. Aconitase is not one of the three rate-determining enzymes of the Citric Acid Cycle as its ΔG is not negative (ΔG°′≈5 kJ/mol and ΔG≈0 kJ/mol).<ref name="Voet" /> Aconitase functions close to equilibrium and the rate of citrate consumption depends on the activity of NAD<sup>+</sup>-dependent isocitrate dehydrogenase, which is one of the three rate-determining enyzmes. Isocitrate dehydrogenase uses the product of the reaction aconitase catalyzes. Both Citrate synthase and Isocitrate dehydogenase are inhibited by NADH concentration, but aconitase itself is not.<ref name="Voet" /> Since the rate of aconitase depends on the activity of NAD<sup>+</sup>-dependent isocitrate dehydrogenase, then citrate could build up on the reactant side, which would then inhibit the enzyme of the previous step, citrate synthase. An illustration of this is seen below, with the boxes representing the enzymes that are catalyzing each reaction. This is a common example of how the Citric Acid Cycle works in order to produce ATP without wasting resources. Similar inhibition/activation of enzymes occurs based on concentrations of ATP, NADH, Calcium, CoA, and others. | |||
[[ Image:Regulation.JPG]] | |||
==Other Functions== | |||
Along with serving as a catalyst, aconitase is a member of the iron regulatory protien-1 (IRP-1) family. These enzymes have been found to play a role in regulatory RNA-binding proteins. This suggests a novel role for Fe-S clusters as post-translational regulatory switches.<ref name="Frishman" /> | |||
==References== | ==References== | ||