Role of key residues of obelin in coelenterazine binding and conversion into 2-hydroperoxy adduct.
Bioluminescence of a variety of marine organisms is caused by monomeric Ca(2+)-regulated photoproteins, to which a peroxy-substituted coelenterazine, 2-hydroperoxycoelenterazine, is firmly bound. From the spatial structure the side chains of Tyr138, His175, Trp179, and Tyr190 of obelin are situated within the substrate-binding pocket at hydrogen bond distances with different atoms of the 2-hydroperoxycoelenterazine. Here we characterized several obelin mutants with substitutions of these residues regarding their bioluminescence, coelenterazine binding, and kinetics of active obelin formation. We demonstrate that Tyr138, His175, Trp179, and Tyr190 are all important for coelenterazine activation; substitution of any of these residues leads to significant decrease of the apparent reaction rate. The hydrogen bond network formed by Tyr138, Trp179 and Tyr190 participates in the proper positioning of coelenterazine in the active site and subsequent stabilization of the 2-hydroperoxy adduct of coelenterazine. His175 might serve as a proton shuttle during 2-hydroperoxycoelenterazine formation.
['Eremeeva|Elena V|EV|', 'Markova|Svetlana V|SV|', 'van Berkel|Willem J H|WJ|', 'Vysotski|Eugene S|ES|']
D019943:Amino Acid Substitution D001059:Apoproteins D002118:Calcium D007093:Imidazoles D007700:Kinetics D008164:Luminescent Proteins D008958:Models, Molecular D009154:Mutation D011485:Protein Binding D011487:Protein Conformation D011719:Pyrazines D013050:Spectrometry, Fluorescence
Obelin Coelenterazine Bioluminescence Photoprotein Aequorin