Sandbox Reserved 895: Difference between revisions

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As stated, the iron ion is also accessible through a main tunnel that enters the helical cap of the protein and is almost orthogonal relative to the propeller axis. This tunnel passes though the metal ion forming a bent cavity within the protein. A secondary tunnel within RPE65 leads from the exterior portion of the protein to the active site but contains a narrow segment that prevents the passage of retinoid substrates as well as other substrates catalyzed by RPE65. Although this second tunnel prevents larger compounds to enter, small molecules like water molecules or ions are allowed into the active site via this tunnel. As such it is hypothesized that the reactants and the products enter and exit from the same tunnel. The presence of amino acid residues such as Phe, Tyr and Trp within the main tunnel help confer enzyme rigidity as well as stabilize the intermediates of the isomerohydrolase reaction. The hydrophobicity of the cavity once again promotes the participation of lipophilic retinoids from the membrane into the active site as well as encourage the reaction of RPE65 with the membrane.

The main tunnel and its interior cavity contain two regions of strong residual electron density that is not accounted for by the atoms located in the protein. This suggest that the substrate would interact with these electron dense regions. The first electron dense region is linear with a triangular shape on one end, suggesting the presence of a linear molecule containing a terminal functional group with trigonal planar geometry (such as an ester in the retinyl esters or a carboxylic acid functional group in fatty acids). The triangular portion is in proximity to the iron ion and when positioned correctly fulfills one or both open coordination sites set forth by the octahedral geometry. The linear portion of the molecule then occupies the main tunnel. The second electron dense region is linear and bent in shape which is located within the interior cavity of the protein. This region could be represented by a bound PEG 200 molecule or a string of partially ordered water molecules. However the electron dense region cannot accommodate longer compound such as (Hydroxyethyloxy)tri(ethyloxy)octane (C8E4).

The main tunnel and its interior cavity contain two regions of strong residual electron density that is not accounted for by the atoms located in the protein. This suggest that the substrate would interact with these electron dense regions. The first electron dense region is linear with a triangular shape on one end, suggesting the presence of a linear molecule containing a terminal functional group with trigonal planar geometry (such as an ester in the retinyl esters or a carboxylic acid functional group in fatty acids). The triangular portion is in proximity to the iron ion and when positioned correctly fulfills one or both open coordination sites set forth by the octahedral geometry. The linear portion of the molecule then occupies the main tunnel. The second electron dense region is linear and bent in shape which is located within the interior cavity of the protein. This region could be represented by a bound PEG 200 molecule or a string of partially ordered water molecules. However, the electron dense region cannot accommodate longer compound such as (Hydroxyethyloxy)tri(ethyloxy)octane (C8E4).

[[Image:RPE65_Binding_Pocket.jpg|thumb|center|512 px|alt=Figure 5: RPE65 Binding Pocket| '''Figure 5:''' RPE65 iron ion binding pocket coordination sites. Shown in olive is the iron ion, shown in red are the bond lengths between the iron atom and the His residues, shown in green are the bond lengths between the His residues and the hydrogen bond interactions with Glu and Val . The bond length interactions are shown in angstroms from the iron atom. <ref> DOI 19805034 </ref>]]

[[Image:RPE65_Binding_Pocket.jpg|thumb|center|512 px|alt=Figure 5: RPE65 Binding Pocket| '''Figure 5:''' RPE65 iron ion binding pocket coordination sites. Shown in olive is the iron ion, shown in red are the bond lengths between the iron atom and the His residues, shown in green are the bond lengths between the His residues and the hydrogen bond interactions with Glu and Val. The bond length interactions are shown in angstroms from the iron atom. <ref> DOI 19805034 </ref>]]

=== ''' [1.2.3] Proposed Enzymatic Mechanism of RPE65''' ===

From an enzymatic point of view, RPE65 is similar to its carotenoid-cleaving relatives in that a ferrous ion is required for catalytic activity. Although the specific enzymatic mechanism for RPE65 is complex and not well elucidated, previous research has shown that the most probable reaction mechanism is as shown in '''Figure 6A''' and '''Figure 6B'''. The initial interaction with the ester moiety of all-trans-retinyl ester with the ferrous ion polarizes the carbon-15 oxygen σ (sigma) bond and makes the fatty ester an activated leaving group. Formation of a resonance-stabilized carbocation reduces the π (pi) bond order of the system allowing temporary rotation about the carbon-11, carbon-12 σ bond. A water molecule obtained from the bulk solution attacks the carbon-15 atom quenching the carbocation intermediate. Finally, the protonation of the fatty acid carboxylate group promotes its dissociation from the ion cofactor. This process releases the fatty acid and the now 11-cis-retinol from the active site of RPE65. <ref> DOI 19805034 </ref>

From an enzymatic point of view, RPE65 is similar to its carotenoid-cleaving relatives in that a ferrous ion is required for catalytic activity. Although the specific enzymatic mechanism for RPE65 is complex and not well elucidated, previous research has shown that the most probable reaction mechanism is shown in '''Figure 6A''' and '''Figure 6B'''. The initial interaction with the ester moiety of all-trans-retinyl ester with the ferrous ion polarizes the carbon-15 oxygen σ (sigma) bond and makes the fatty ester an activated leaving group. Formation of a resonance-stabilized carbocation reduces the π (pi) bond order of the system allowing temporary rotation about the carbon-11, carbon-12 σ bond. A water molecule obtained from the bulk solution attacks the carbon-15 atom quenching the carbocation intermediate. Finally, the protonation of the fatty acid carboxylate group promotes its dissociation from the ion cofactor. This process releases the fatty acid and the now 11-''cis''-retinol from the active site of RPE65. <ref> DOI 19805034 </ref>

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@@ Line 52: / Line 52: @@
 As stated, the iron ion is also accessible through a main tunnel that enters the helical cap of the protein and is almost orthogonal relative to the propeller axis. This tunnel passes though the metal ion forming a bent cavity within the protein. A secondary tunnel within RPE65 leads from the exterior portion of the protein to the active site but contains a narrow segment that prevents the passage of retinoid substrates as well as other substrates catalyzed by RPE65. Although this second tunnel prevents larger compounds to enter, small molecules like water molecules or ions are allowed into the active site via this tunnel. As such it is hypothesized that the reactants and the products enter and exit from the same tunnel. The presence of amino acid residues such as Phe, Tyr and Trp within the main tunnel help confer enzyme rigidity as well as stabilize the intermediates of the isomerohydrolase reaction. The hydrophobicity of the cavity once again promotes the participation of lipophilic retinoids from the membrane into the active site as well as encourage the reaction of RPE65 with the membrane.
-The main tunnel and its interior cavity contain two regions of strong residual electron density that is not accounted for by the atoms located in the protein. This suggest that the substrate would interact with these electron dense regions. The first electron dense region is linear with a triangular shape on one end, suggesting the presence of a linear molecule containing a terminal functional group with trigonal planar geometry (such as an ester in the retinyl esters or a carboxylic acid functional group in fatty acids). The triangular portion is in proximity to the iron ion and when positioned correctly fulfills one or both open coordination sites set forth by the octahedral geometry. The linear portion of the molecule then occupies the main tunnel. The second electron dense region is linear and bent in shape which is located within the interior cavity of the protein. This region could be represented by a bound PEG 200 molecule or a string of partially ordered water molecules. However the electron dense region cannot accommodate longer compound such as (Hydroxyethyloxy)tri(ethyloxy)octane (C8E4).
+The main tunnel and its interior cavity contain two regions of strong residual electron density that is not accounted for by the atoms located in the protein. This suggest that the substrate would interact with these electron dense regions. The first electron dense region is linear with a triangular shape on one end, suggesting the presence of a linear molecule containing a terminal functional group with trigonal planar geometry (such as an ester in the retinyl esters or a carboxylic acid functional group in fatty acids). The triangular portion is in proximity to the iron ion and when positioned correctly fulfills one or both open coordination sites set forth by the octahedral geometry. The linear portion of the molecule then occupies the main tunnel. The second electron dense region is linear and bent in shape which is located within the interior cavity of the protein. This region could be represented by a bound PEG 200 molecule or a string of partially ordered water molecules. However, the electron dense region cannot accommodate longer compound such as (Hydroxyethyloxy)tri(ethyloxy)octane (C8E4).
-[[Image:RPE65_Binding_Pocket.jpg|thumb|center|512 px|alt=Figure 5: RPE65 Binding Pocket| '''Figure 5:''' RPE65 iron ion binding pocket coordination sites. Shown in olive is the iron ion, shown in red are the bond lengths between the iron atom and the His residues, shown in green are the bond lengths between the His residues and the hydrogen bond interactions with Glu and Val . The bond length interactions are shown in angstroms from the iron atom. <ref> DOI 19805034 </ref>]]
+[[Image:RPE65_Binding_Pocket.jpg|thumb|center|512 px|alt=Figure 5: RPE65 Binding Pocket| '''Figure 5:''' RPE65 iron ion binding pocket coordination sites. Shown in olive is the iron ion, shown in red are the bond lengths between the iron atom and the His residues, shown in green are the bond lengths between the His residues and the hydrogen bond interactions with Glu and Val. The bond length interactions are shown in angstroms from the iron atom. <ref> DOI 19805034 </ref>]]
 === ''' [1.2.3] Proposed Enzymatic Mechanism of RPE65''' ===
-From an enzymatic point of view, RPE65 is similar to its carotenoid-cleaving relatives in that a ferrous ion is required for catalytic activity. Although the specific enzymatic mechanism for RPE65 is complex and not well elucidated, previous research has shown that the most probable reaction mechanism is as shown in '''Figure 6A''' and '''Figure 6B'''. The initial interaction with the ester moiety of all-trans-retinyl ester with the ferrous ion polarizes the carbon-15 oxygen σ (sigma) bond and makes the fatty ester an activated leaving group. Formation of a resonance-stabilized carbocation reduces the π (pi) bond order of the system allowing temporary rotation about the carbon-11, carbon-12 σ bond. A water molecule obtained from the bulk solution attacks the carbon-15 atom quenching the carbocation intermediate. Finally, the protonation of the fatty acid carboxylate group promotes its dissociation from the ion cofactor. This process releases the fatty acid and the now 11-cis-retinol from the active site of RPE65. <ref> DOI 19805034 </ref>
+From an enzymatic point of view, RPE65 is similar to its carotenoid-cleaving relatives in that a ferrous ion is required for catalytic activity. Although the specific enzymatic mechanism for RPE65 is complex and not well elucidated, previous research has shown that the most probable reaction mechanism is shown in '''Figure 6A''' and '''Figure 6B'''. The initial interaction with the ester moiety of all-trans-retinyl ester with the ferrous ion polarizes the carbon-15 oxygen σ (sigma) bond and makes the fatty ester an activated leaving group. Formation of a resonance-stabilized carbocation reduces the π (pi) bond order of the system allowing temporary rotation about the carbon-11, carbon-12 σ bond. A water molecule obtained from the bulk solution attacks the carbon-15 atom quenching the carbocation intermediate. Finally, the protonation of the fatty acid carboxylate group promotes its dissociation from the ion cofactor. This process releases the fatty acid and the now 11-''cis''-retinol from the active site of RPE65. <ref> DOI 19805034 </ref>
 [[Image:RPE65_Figure6_Reaction_mechanism.jpg|thumb|center|512 px|alt=Figure 6: RPE65 Hypothesized Reaction Mechanism| '''Figure 6:''' (A) All-''trans''-retinol (B) Hypothesized RPE65 Reaction Mechanism <ref> DOI 19805034 </ref>]]