Characterization of the binding of Photobacterium phosphoreum P-flavin by Vibrio harveyi Luciferase.
The isolated Photobacterium phosphoreum luciferase is associated with a bound flavin designated P-flavin and tentatively identified as 6-(3"-myristic acid)-FMN. Since FMN and myristic acid are products of the normal luciferase reaction, we explored the possibility that P-flavin can also be bound by luciferase from other luminous bacteria and serve as an active site probe. P-flavin has never been detected in Vibrio harveyi cells. We found that the V. harveyi luciferase binds P. phosphoreum P-flavin, at a ratio of 1 P-flavin per luciferase alphabeta dimer, and with concomitant absorption spectral perturbation of P-flavin, fluorescence quenching of P-flavin and luciferase, and activity inhibition of luciferase. Isolated P-flavin can be fully reduced photochemically. V. harveyi luciferase bound the oxidized P-flavin with a K(d) (or K(i) competitively against decanal) of 0.1-0.16 microM, which is three orders of magnitude lower than the K(d) for FMN binding but similar to that of reduced FMN binding. The reduced P-flavin exhibited a K(i) (competitively against the reduced FMN substrate) of 0.16 microM, also similar to the K(d) for reduced FMN. Hence, the covalent attachment of myristic acid to FMN greatly and preferentially enhanced the binding of oxidized P-flavin. The dissociation of P-flavin was slow in comparison with the binding of reduced FMN and decanal substrates. Modification of the alphaCys106 near the active site by N-ethylmaleimide can be retarded by P-flavin. These findings indicate that P-flavin is potentially a superb active site probe for luciferase. We hypothesize that P-flavin is a by-product of luciferase generated by a side reaction which is trivial with the V. harveyi luciferase but significant in the P. phosphoreum luciferase-catalyzed reaction.
Wei
C J
CJ
Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001, USA.
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D001665:Binding Sites; D001667:Binding, Competitive; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry; D005033:Ethylmaleimide / Q000494:pharmacology; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism; D010776:Photobacterium / Q000737:chemistry*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014733:Vibrio / Q000201:enzymology*
D005415:Flavins; D008156:Luciferases; D003545:Cysteine; D005033:Ethylmaleimide