Isolation and characterization of the transient, luciferase-bound flavin-4a-hydroxide in the bacterial luciferase reaction
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چکیده
Procedures and conditions have been established such that the unstable enzyme-bound flavin intermediate produced in the bacterial luciferase reacti~ can be isolated as approximately 70% of the flavin product, the remaining being the final product, FMN. The structure of the intermediate is proposed to be that of a luciferase-bound 4a,5-dihydroflavin-4a-hydroxide. The intermediate has a half-life of 33 min at 2° C and decays spontaneously to give H 20 and luciferase-bound FMN with an activation enthalpy of about 120 kJ Imo!. It has an absorption spectrum (A max = 360 nm) that is consistent with the proposed structure, and a fluorescence emission (A max =485 nm) that matches the bioluminescence emission closely.
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