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== Structure == | == Structure == | ||
[[Image:homotetramer.png|150 px|left|thumb|Figure 2: 222 Symmetry of the homotetramer isocitrate lyase. Each identical monomer is shown in a unique color]] | [[Image:homotetramer.png|150 px|left|thumb|Figure 2: 222 Symmetry of the homotetramer isocitrate lyase. Each identical monomer is shown in a unique color.]] | ||
The ICL homotetramer possesses 222 symmetry, with an axis of rotation at x-axis, y-axis, and z-axis of the enzyme. Two individual subunits of ICL are held together by a characteristic <scene name='69/697526/Helix_swapping/3'>Helix Swapping</scene> between three alpha helices formed by residues 370-384, 349-367, and 399-409 on neighboring monomers<ref name="ICL">PMID:10932251</ref>. The interlocking mechanism created by these helices provides additional strength to hold the two monomeric subunits together, allowing ICL to be composed of a dimer of dimers<ref name="ICL2"/>. This interaction will bury approximately 18% of the surface of each subunit, and will help to shield the interior binding site from hydration. | The ICL homotetramer possesses 222 symmetry, with an axis of rotation at x-axis, y-axis, and z-axis of the enzyme. Two individual subunits of ICL are held together by a characteristic <scene name='69/697526/Helix_swapping/3'>Helix Swapping</scene> between three alpha helices formed by residues 370-384, 349-367, and 399-409 on neighboring monomers<ref name="ICL">PMID:10932251</ref>. The interlocking mechanism created by these helices provides additional strength to hold the two monomeric subunits together, allowing ICL to be composed of a dimer of dimers<ref name="ICL2"/>. This interaction will bury approximately 18% of the surface of each subunit, and will help to shield the interior binding site from hydration. | ||
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===Catalytic Loop=== | ===Catalytic Loop=== | ||
[[Image:Openvsclosed..png|400 px|left|thumb|Figure 4: Open (Blue) vs. Closed (Red) conformation of the active site loop of ICL. Glyoxylate is shown in yellow and succinate in purple. Hydrogen | [[Image:Openvsclosed..png|400 px|left|thumb|Figure 4: Open (Blue) vs. Closed (Red) conformation of the active site loop of ICL. Glyoxylate is shown in yellow and succinate in purple. Hydrogen bonding is shown between LYS189 and the catalytic loop.]] | ||
The <scene name='69/694223/Catalytic_loop/2'>Catalytic Loop</scene> of the Isocitrate Lyase enzyme is composed of residues 185-196, and can exist in both the open and closed conformation. In the open conformation, the catalytic loop is oriented such that the catalytic CYS191 residue is located far from the active site, allowing for solvent accessibility and substrate binding.<ref name="solvent">Connely, M. L. Solvent-accessible surfaces of proteins and nucleic acids "Science" 221:709-713 (1983). DOI: 10.1126/science.6879170</ref> Upon substrate binding, the catalytic loop adopts a closed conformation, moving between ten and fifteen angstroms<ref name="ICL">PMID:10932251</ref>. This closed conformation will cause the binding site to become inaccessible to the solvent. The loop closure is triggered by the movement of the Mg ion that occurs upon binding of the succinate. This movement of the Mg ion results in electrostatic interactions at LYS189, causing the loop to close. | The <scene name='69/694223/Catalytic_loop/2'>Catalytic Loop</scene> of the Isocitrate Lyase enzyme is composed of residues 185-196, and can exist in both the open and closed conformation. In the open conformation, the catalytic loop is oriented such that the catalytic CYS191 residue is located far from the active site, allowing for solvent accessibility and substrate binding.<ref name="solvent">Connely, M. L. Solvent-accessible surfaces of proteins and nucleic acids "Science" 221:709-713 (1983). DOI: 10.1126/science.6879170</ref> Upon substrate binding, the catalytic loop adopts a closed conformation, moving between ten and fifteen angstroms<ref name="ICL">PMID:10932251</ref>. This closed conformation will cause the binding site to become inaccessible to the solvent. The loop closure is triggered by the movement of the Mg ion that occurs upon binding of the succinate. This movement of the Mg ion results in electrostatic interactions at LYS189, causing the loop to close. | ||
==Elucidation of ICL Structure Using Inhibitors== | ==Elucidation of ICL Structure Using Inhibitors== | ||
[[Image:Inhibitors57.png|400 px|right|thumb|Figure 5: Substrate inhibitors of Isocitrate Lyase]] | [[Image:Inhibitors57.png|400 px|right|thumb|Figure 5: Substrate inhibitors of Isocitrate Lyase used to elucidate the first crystal structure of ICL.]] | ||
The two inhibitors used to elucidate the structure of ICL were 3-nitropropionate and 3-bromopyruvate. The 3-nitropropionate was used to mimic the succinate, while the 3-bromopyruvate is used to mimic the glyoxylate. These two inhibitors have also been shown to be good inhibitors of isocitrate lyase in ''M. avium'' indicating that their inhibitory capacity is conserved across multiple species<ref name="ICL">PMID:10932251</ref>. A mutant isocitrate lyase C191S, in conjunction with the aforementioned substrate mimics, was used to elucidate the first high resolution crystal structure of ICL <ref name="ICL">PMID:10932251</ref>. Dehalogenated 3-bromopyruvate works to inhibit isocitrate lyase by forming a covalent bond with the Ser191 in the active site, which can be found crystallized here [[1f8m]]. This 3-bromopyruvate occupies the same site that the succinate would occupy. The C191S mutant adopts a conformation almost identical to the CYS191 residue in the wild type indicating that this is an accurate depiction of the conformation <ref name="ICL">PMID:10932251</ref>. | The two inhibitors used to elucidate the structure of ICL were 3-nitropropionate and 3-bromopyruvate. The 3-nitropropionate was used to mimic the succinate, while the 3-bromopyruvate is used to mimic the glyoxylate. These two inhibitors have also been shown to be good inhibitors of isocitrate lyase in ''M. avium'' indicating that their inhibitory capacity is conserved across multiple species<ref name="ICL">PMID:10932251</ref>. A mutant isocitrate lyase C191S, in conjunction with the aforementioned substrate mimics, was used to elucidate the first high resolution crystal structure of ICL <ref name="ICL">PMID:10932251</ref>. Dehalogenated 3-bromopyruvate works to inhibit isocitrate lyase by forming a covalent bond with the Ser191 in the active site, which can be found crystallized here [[1f8m]]. This 3-bromopyruvate occupies the same site that the succinate would occupy. The C191S mutant adopts a conformation almost identical to the CYS191 residue in the wild type indicating that this is an accurate depiction of the conformation <ref name="ICL">PMID:10932251</ref>. | ||