Fumarase 2: Difference between revisions

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===Stucture===

===Stucture: will the real active site please stand?===

Fumarase is classified as an all alpha protein which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits. 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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The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits and is located in a relatively deep pit that is removed from bulk solvent. The multi-subunit active site is comprised of atoms from residues 312-334 from subunit A, residues 182-200 from subunit C, and residues 129-145 from subunit D<ref>PMID: 7552727</ref>. The residues that form the active site are N141b, T100b, S98b, E331c, K324c, N326c, His 188C, and a water molecule, W-26. It is speculated that the H188 and W-26 are two of the most vital active site residues. Furthermore, H188 and W-26 form a short hydrogen bond, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of <scene name='72/726367/L-malate/1'>L-malate</scene>. The cationic charge on W-26 plays an essential role in the stabilization of the double negative charge that is present on the aci-carboxylate at C4. Complex hydrogen bonding patterns in the active site involving T187, N141, H188, N362, and K324 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.

~~===Regulation===~~

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.

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.

Fumarase 2: Difference between revisions

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@@ Line 6: / Line 6: @@
-===Stucture===
+===Stucture:  will the real active site please stand?===
 Fumarase is classified as an all alpha protein which belongs to the L-aspartase/fumarase family. It forms a tetramer of identical subunits.  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?
@@ Line 14: / Line 14: @@
 The active site (A-site) of the fumarase enzyme is formed by residues from three of the enzyme’s four subunits and is located in a relatively deep pit that is removed from bulk solvent. The multi-subunit active site is comprised of atoms from residues 312-334 from subunit A, residues 182-200 from subunit C, and residues 129-145 from subunit D<ref>PMID: 7552727</ref>. The residues that form the active site are N141b, T100b, S98b, E331c, K324c, N326c, His 188C, and a water molecule, W-26. It is speculated that the H188 and W-26 are two of the most vital active site residues. Furthermore, H188 and W-26 form a short hydrogen bond, which increases the basicity of the water molecule. This electron-withdrawing hydrogen bond allows the water molecule to remove the C3 proton of <scene name='72/726367/L-malate/1'>L-malate</scene>. The cationic charge on W-26 plays an essential role in the stabilization of the double negative charge that is present on the aci-carboxylate at C4. Complex hydrogen bonding patterns in the active site involving T187, N141, H188, N362, and K324 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.
-===Regulation===
-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.
-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.