title: The active-site environment of rhodopsin.
abstract: The 11-cis-retinal binding site of rhodopsin is of great interest because it is buried in the membrane but yet must provide an environment for charged amino acids. In addition, the active-site lysine residue must be able to engage in rapid Schiff base formation with 11-cis-retinal at neutral and lower pH values. This requires that this lysine be unprotonated. We have begun to study the environment of the active-site lysine using a reporter group adducted to it. Non-active-site permethylated opsin was reacted with 5-nitrosalicylaldehyde, and the resulting Schiff base was permanently fixed by borohydride reduction. The stoichiometry of incorporation was one. This chromophoric and pH-sensitive reporter group affords information on the active-site environment of rhodopsin by determining the ionization constants of its ionizable groups at different pH values. The pH titration of the modified protein showed a single pKa = 7.8 +/- 0.19 ascribable to the ionization of the phenol. The ionization of the modified lysine residue was not observed at all pH values studied. These studies are interpreted to mean that a negatively charged amino acid is propinquous to the active-site lysine residue and that this latter residue does not have an unusually low pKa.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: M A
authors.initials: MA
authors.lastname: Pajares
authors.forename: R R
authors.initials: RR
authors.lastname: Rando
mesh_terms: D000447:Aldehydes; D000818:Animals; D001547:Benzaldehydes; D001665:Binding Sites; D002417:Cattle; D005136:Eye Proteins / Q000648:ultrastructure*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008745:Methylation; D012168:Retinal Pigments; D012243:Rhodopsin / Q000648:ultrastructure*; D012374:Rod Cell Outer Segment; D017299:Rod Opsins; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000447:Aldehydes; D001547:Benzaldehydes; D005136:Eye Proteins; D012168:Retinal Pigments; D017299:Rod Opsins; D012243:Rhodopsin; C015393:5-nitrosalicylaldehyde