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Q. Yuan, M. Jin, S. Li, G. H. Travis; Reaction Mechanism of Rpe65-isomerase Studied Using a Suicide Inhibitor and Mass Spectrometry. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2967.
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The enzymatic conversion of an all-trans-retinyl ester to 11-cis-retinol (11-cis-ROL) is a critical step in the regeneration of visual chromophore, and is catalyzed by the recently identified Rpe65-isomerase. The biochemical mechanism of this reaction is unknown. Rpe65 is homologous to apo-carotene oxygenase (ACO), whose structure was recently solved. Both proteins comprise a seven-bladed ß-propeller with a Fe2+-4-His sphere at its axis. Iron-binding and the presence of these His residues are required for Rpe65-isomerase activity. Based on the predicted structure and biochemical properties of Rpe65, we propose a mechanism for the isomerase reaction.
Indicated residues in bovine Rpe65 were substituted by site-directed mutagenesis. Wild-type and substituted Rpe65’s were expressed in 293T or 293T-LC cells that stably express LRAT and CRALBP. Isomerase activity was measured by monitoring formation of 11-cis-ROL from all-trans-retinyl palmitate (all-trans-RP) added to cell homogenates or the culture media. A suicide inhibitor designed according to our hypothesized reaction mechanism was synthesized chemically. This inhibitor was added to homogenates of Rpe65-expressing cells or bovine retinal pigment epithelium (RPE) to verify suppression of isomerase activity. Purified Rpe65 from bovine RPE was incubated with the inhibitor and analyzed by mass spectrometry following proteolytic digestion. Mass spectrometry has been using to identify the peptide moiety involving in the isomerization.
Wild-type Rpe65 synthesized significant 11-cis-ROL from all-trans-ROL in 293T-LC cells. However, Rpe65’s with substitutions in residues at the proposed catalytic site exhibited no isomerase activity. Synthesis of 11-cis-ROL from all-trans-RP by Rpe65 in bovine RPE homogenates was significantly reduced in the presence of the suicide inhibitor.
Loss of catalytic activity with substitution of a proposed critical residue within the catalytic cleft suggests that this residue plays a role in the reaction mechanism. Isomerase inhibition by the suicide inhibitor also supports our hypothesized mechanism.
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