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| - | <StructureSection load='1fuo' size='350' side='right' caption='Fumarase with citrate bound to the active site (PDB profile: 1fuo)'> | + | ==Fumarase== |
| - | =Fumarase= | + | <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=== | | ===Overview=== |
| - | Fumarase, also known as fumarate hydratase, is an enzyme in the Krebs cycle, also known as 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''', 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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| - | ===Stucture===
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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 symmetrical tetramer. Within each subunit, 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 to the substrate, the B site shifts substantially more upon binding, and this shift helps regulate affinity for molecule binding at the active site <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>. This has several implications for regulation of fumarase's activity and affinity to bind at the active site, but water molecules also play an important role in its function as an enzyme. | + | |
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| - | ===Mechanism of Reaction===
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| - | The reaction mechanism consists of two main steps. The first step of the reaction involves addition of a hydroxy group from the water molecule to a double-bonded carbon in fumarate. When the hydroxy ion bonds with a carbon, an electron from the double bond moves to the other carbon atom which forms a carbanion transition state. Finally, a proton from the water molecule bonds to the carbanion, forming malate <ref>Voet, D., Voet, J. & Pratt, C. ''Fundamentals of Biochemistry: Life at the Molecular Level''. 3rd Ed. NJ: John Wiley & Sons, Inc., (2008), 583.</ref>. In the <scene name='Vas_Sandbox_1/Active_site/1'>active site</scene>, amino acid residues involved in binding the substrate are located on three subunits: Thr100, Ser139, Ser140, and Asn141 from the b-subunit, Thr187 and His188 on the d-subunit, and Lys324 and Asn326 of the c-subunit <ref name="Beeckmans, et al."> Beeckmans, S. & Van Driessche, E. Pig heart fumarase contains two distinct substrate-binding sites differing in affinity. ''Journal of Biological Chemistry'' (1998), '''273'''(48), 31661-31669.</ref>. The B site is located in a π-helix turn between the active site and solvent, and it includes residues Arg126, Lys127, Val128, His129, Pro130, Asn131, and Asp132 all on the b-subunit. Two hydrogen bonds initiate the binding of Asn131 and Asp132 residues with S-malate.
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| - | ===Kinetics===
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| - | Fumarase catalyzes the reversible reaction between fumarate and malate in the citric acid cycle of cellular metabolism. When it catalyzes the addition of water to fumarate in order to form malate, the Km and Vmax values are 0.30 mM and 129 s<sup>-1</sup>, respectively. The reverse reaction (dehydration of malate to form fumarate) has a Km of 0.10 mM and Vmax of 60 s<sup>-1</sup> <ref name="Rose & Weaver"> Rose, I. & Weaver, T. The role of the allosteric B site in the fumarase reaction. ''Proc. Natl. Acad. Sci. USA'' (2004), '''101'''(10), 3393-3397. ['''http://www.pnas.org/cgi/doi/10.1073/pnas.0307524101''' doi:10.1073/pnas.0307524101]</ref>. Thus, the forward pathway is favored because of its higher Km and Vmax values which means that converting fumarate to malate is more energetically favorable. Fumarase kinetics normally follows Michaelis-Menten kinetic plots at low concentrations of substrate, but high substrate concentrations influence the enzyme’s activity due to allosteric effects. 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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| - | ===Regulation===
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| - | Similar to most enzymes involved in biological processes, fumarase can be regulated in several different ways. Allosteric effects commonly regulate fumarase activity via substrate and inhibitor binding with the active site. Fumarase activity is both positively and negatively regulated by substrate concentration. When the concentration of a substrate is five-fold of the Km value, it activates fumarase’s activity; however, substrate concentrations above 0.1 M result in inhibition of fumarase function <ref name="Beeckmans, et al."/>. The concentration of substrate influences cooperativity of fumarase, depending on the availability of substrate to bind to domains.
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| - | The regulation of fumarase via allosteric effects involves conformational changes that occur when a substrate binds to the active site. Studies involving amino acid residue manipulation in the B site show that the B site helps regulate the binding affinity for the active site by allosteric effects <ref name="Beeckmans, et al."/>,<ref name="Rose & Weaver"/>. According to Weaver (2004), the active site and B site are located 12 Å apart which suggests that the conformational changes resulting from <scene name='Vas_Sandbox_1/Malate_interaction/2'>malate interactions</scene> with the B site influence the active site affinity to bind with the substrate <ref name="Weaver, et al."/>. Inhibitors also regulate the activity of an enzyme via binding to the active site. Both citrate and succinate are known as competitive inhibitors of fumarase since they negatively influence the enzyme’s activity. They are competitive inhibitors because they have structural similarity to the substrate; therefore, the inhibitors compete with substrates to bind with the active site. The natural state of fumarase commonly involves <scene name='Vas_Sandbox_1/Citrate_interaction/2'>citrate interactions</scene> with the active site in which similar amino acid residues responsible for binding with a substrate result in binding with a citrate molecule.
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| | + | ==Structure: will the real active site please stand?== |
| | + | 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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| - | ===Other Interesting Information=== | + | 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>. |
| - | Fumarase expression mainly occurs in skin, parathyroid, lymph, and colon tissues, and it is present throughout all life stages, from early development to mature adults. Fumarase comprises two specific classes which relate to the enzyme's: arrangement of subunits, metal ion requirement, and thermal stability. Class I fumarase isozymes can change their state, become inactive upon exposure to heat or radiation, are sensitive to superoxide anions, and Fe2+ dependent. Class II includes fumarase found in eukaryotes and prokaryotes, and they are iron-independent and thermally stable.
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| - | Mutations in the gene that encodes fumarase can lead to a deficiency in fumarase enzyme in the citric acid cycle which is known to cause certain diseases. Autosomal recessive mutants can result in fumarase deficiency, a metabolic disorder distinguished by excess fumarate in the body which can lead to severe developmental defects <ref>Remes, A., Rantala, H., Hiltunin, K., Leisti, J. & Ruokonen, A. Fumarase deficiency: two siblings with enlarged cerebral ventricles and polyhydramnios in utero. ''Pediatrics'' (1992), '''89'''(4), 730-734.</ref>. Inheritance of this autosomal recessive mutation has serious effects on early neural and brain development and can be fatal. Also, heterozygous fumarase mutations play a role in cancerous tumor development; specifically, the mutant H153R has identified as a factor in three families of malignant tumor growths <ref>Kokko, A., Ylisaukko-Oja, S., Kiuru, M., Takatalo, M., Salmikangas, P., Tuimala, J., et al. Modeling tumor predisposing FH mutations in yeast: effects on fumarase activity, growth phenotype and gene expression profile. ''Int. J. Cancer'' (2006), '''118'''(6), 1340-1345.</ref>.
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| - | </structure section> | + | == 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> |
| | ===References=== | | ===References=== |
| | <references/> | | <references/> |
