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| ==This is a placeholder== | | ==Fumarase== |
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| | <StructureSection load='1fuo' size='340' side='right' caption='Fumarase with citrate bound to the active site (PDB profile: 1fuo)' scene = ''> |
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| | ===Overview=== |
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| | '''Fumarase''', also known as fumarate hydratase, is an enzyme in the citric acid cycle. In the seventh step of the reaction pathway, fumarase catalyzes the reversible hydration reaction that converts fumarate to malate and vice versa. Fumarase is classified as an <scene name='44/446278/Secondary_structure/2'>alpha helical protein</scene> which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits that <scene name='44/446278/Rainbow_subunits/1'>alternate in orientation</scene>. Each subunit is comprised of <scene name='44/446278/Domains/1'>three domains</scene>. |
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| | ==Structure: will the real active site please stand?== |
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| | Crystal structures of fumarase C revealed that the enzyme has two dicarboxylate binding sites; one was called the A site, and the second, the B site. This raises the question: which of the two sites is the active site of the enzyme? The A site shows relatively little change upon substrate binding, while the B site shifts substantially. <ref name="Weaver, et al."> Weaver,T. Structure of free fumarase C from ''Escherichia coli''. ''Acta Crystallographica'' (2005), '''D61''', 1395-1401. ['''http://dx.doi.org/10.1107/S0907444905024194''' doi:10.1107/S0907444905024194]</ref>. But these changes could account for regulation...so which site is the true active site? |
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| {{STRUCTURE_3cin | PDB=3cin | SCENE= }}
| | In order to answer this question, an experiment that tested each of the sites independently was conducted. Both sites contain histidine residues: <scene name='44/446278/His_188/1'>His 188</scene> in the A-site and <scene name='44/446278/His_129/1'>His 129</scene> in the B-site. These sites were mutated to asparagine in separate experiments, and the effect on kinetics was measured. The results of the experiment showed that the H129N mutation had little effect on the enzymatic activity of the enzyme, as the specific activity of the enzyme was comparable to the wild-type enzyme. In contrast, the <scene name='72/726367/Ans_188_mutant/1'>H188N</scene> mutation dramatically reduced the specific activity of the catalytic reaction. These data strongly suggested that the H188 residue had a direct role in the catalytic mechanism of the enzyme and, therefore, that the H188 residue was located in the active site of the enzyme. This lead to the conclusion that that the A-site was in fact the active site of the enzyme<ref name= "Weaver">PMID:9098893</ref>. |
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| =Fumarase= | | == Active Site Characteristics == |
| | | The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits (shown in <scene name='44/446278/Active_site_chains/3'>different colors</scene>) and is located in a relatively deep pit that is removed from bulk solvent <ref>PMID: 7552727</ref>. The residues that form the <scene name='44/446278/Active_site_residues/6'>active site</scene> include N141b, T100b, S98b, E331c, K324c, N326c, His 188c, (the letter indicates the chain) and a water molecule. It is speculated that the <scene name='44/446278/His_188_active_site/2'>H188</scene> is the most important active site residue, activating the water through a <scene name='44/446278/Short_h_bond/2'>short hydrogen bond</scene>, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of malate, though this model has <scene name='44/446278/Citrate/2'>citrate</scene> in the active site. Complex hydrogen bonding patterns in the active site also help stabilize the aci-carboxylate intermediate<ref name= "Weaver">PMID:9098893</ref>. By increasing the stabilization if the intermediate, the fumarase enzyme can effectively catalyze the hydration/dehydration reaction between L-malate and fumarate. |
| | | </StructureSection> |
| == Overview ==
| | ===References=== |
| Fumarase, also known as fumarate hydratase, functions as an enzyme in the metabolic pathway known as the Kreb’s cycle, or citric acid cycle. As the seventh step in the pathway, fumarase catalyzes the reversible reaction converting fumarate to S-malate. It metabolizes Fumarate in the cytosol, which becomes a byproduct of the urea cycle and amino acid catabolism. It catalyzes the addition of water to make S-Malate; therefore, the mechanism of fumarase in the reaction involves hydration of fumarate in order to form S-malate.
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| == Stucture and Classification ==
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| Fumarase is classified as an all alpha protein which belongs to the L-aspartase/fumarase family, and the enzyme specifically consists of four identical subunits which form a tetramer. From the four subunits, fumarase has three domains which comprise two binding sites: the active site and B site. Although the active site has a mostly solid structure and shifts very little when it binds, the B site shifts substantially more upon binding, and this shift helps regulate affinity for molecule binding at the active site (Weaver 2005).
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| ==Mechanism of Reaction==
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| Fumarase has the ability to catalyze the hydration of fumarate to malate or the dehydration of malate to fumarate. The mechanism of fumarase in the hydration and dehydration reaction pathways remains simple, only involving three steps. In the dehydration reaction, fumarase deprotonates a carbon atom on malate to form a carbanion (Beechmans 1998). This deprotonation results in an aci-carboxylate intermediate (Weaver 2005). After the intermediate forms, the acidic proton from the initial step removes the hydroxide group from the aci-carboxylate intermediate to form fumarase which then detaches from the active site of fumarase, completing the reaction (Rose, Weaver, 2004). The active site binds with the substrate via Asn326 and Lys324 residues which function in the binding process. The B site also can bind with S-malate, and the residues His129, Asn131, Asp132, and Arg126 contribute to binding (Weaver 2005). The B site is located in a π-helix turn between the active site and solvent. Two hydrogen bonds initiate the binding of Asn131 and Asp132 residues with S-malate (Rose, Weaver, 2004).
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| ==Enzyme Kinetics==
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| Fumarase catalyzes the reversible reaction between S-malate and fumarate in the citric acid cycle for cellular metabolism. When it catalyzes the addition of water to S-malate in order to form fumarate, the Km and Vmax values are 0.30 mM and 129 s^-1, respectively. The reverse reaction (dehydration of fumarate to form S-malate) has a Km of 0.10 mM and Vmax of 60 s^-1 (Rose, Weaver 2004). Thus, the forward pathway (fumarate to S-malate) is favored because of its higher Km and Vmax values. According to Beechmans (1998), fumarase kinetics normally follows Michaelis-Menten kinetic plots with low concentrations of substrate, but high concentrations influence the enzyme’s activity. Enzyme kinetics studies with fumarase mutants differ from wild type fumarase kinetics when the mutations alter amino acid residues involved in binding at the active site and B site. Also, the Michaelis-Menten kinetics plots for fumarase mutants exhibit a sigmoidal curve which suggests the presence of cooperativity in the enzymes activity.
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| Name of enzyme: Fumarase or Fumarate hydratase
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| Pathway and reaction catalyzed with metabolite structures: Fumarase is used in the citric acid cycle to conduct a transition step in the production of energy to make NADH. This is a reversible reaction. Conversion of Fumarate to S-Malate using Fumarase: Image:400px-Reaction1.png
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| Other interesting information: Fumarase is dominant in fetal and adult tissues and largely expressed in the skin, parathyroid, lymph, and colon There are two classes of Fumarases, which depend on the arrangement of their relative subunit, their metal requirement, and their thermal stability. Class I Fumarases can change their state or become inactive when exposed to heat or radiation. They are sensitive to superoxide anions and Fe2+ dependent. Class II Fumarases are found in eukaryotes and prokaryotes. They are iron-independent and thermal-stable. Fumarase deficiency is an autosomal recessive metabolic disorder distinguished by a deficiency of the enzyme Fumarate hydratase and indicated by an excess of Fumaric acid in the urine. It is common of infants with neurologic abnormalities and its potential causes include cytosolic and mitochondrial forms of Fumarase.
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