Sandbox Reserved 895: Difference between revisions

Like stated before the RPE65 iron cofactor is found near the top surface of the β-propeller structure. The iron ion is directly coordinated by the Nitrogen-ε atoms of His180, His241, His313 and His527 with an average bond length of 2.2 angstroms. The geometry of the iron ion coordination is that of an octahedral with two open coordination sties. With the exception of His180, the other coordination interactions (His241, His313 and His527) is via hydrogen bonding from the Nδ-H bond on histidine to the Oε on Glu148, Glu417 and Glu469 respectively. The Nδ-H bond on His180 coordinates with a water molecule that is found lower axial, in the hydrophilic region of the propeller. These hydrogen bonds stabilize the octahedral coordination of the iron ion to facilitate the catalysis reaction. Of the two open coordination sties, one of the sites is occupied by the Cγ atom from Val134 located approximately 4.9 angstroms away. These interactions are shown in '''Figure 5A'''.  

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 show in '''Figure 4C'''. 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 occludes the passage of retinoid substrates as well as other substrates catalyzed by RPE65. Although this second tunnel occludes larger compounds, water, small molecules and ions are still permitted 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 isomerhydrolase 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. Shown in '''Figure 5B''', 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 while the linear portion occupies the main tunnel. Also shown in '''Figure 5B''', 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 molecule. However the electron dese region cannot accommodate an longer compound such as (Hydroxyethyloxy)tri(ethyloxy)octane (C8E4).  

[[Image:RPE65_Figure5_Binding_Pocket.jpg|thumb|center|512 px|alt=Figure 5: RPE65 Binding Pocket| '''Figure 5:''' RPE65 iron ion binding pocket coordination sites (A) Stereoview of the iron cofactor and its ligand. Shown in orange is the iron ion, shown in red is a water molecule. The bond length interactions are shown in angstroms from the iron atom. (B) Stereoview of the residual electron density from the RPE65 active site. The green mesh represents an unbiased electron density map contoured at 3.5 σ. The numbers next to teh dash lines indicates bond length interactions measured in angstroms. <ref> DOI 19805034 </ref>]]