Binding Site Determination from Kinetic Data

The elucidation of the number of reduced flavin mononucleotide (FMNH2)-binding sites in bacterial luciferase is of fundamental importance for understanding the mechanism of the bioluminescent reaction. At present, knowledge concerning the binding sites is meager, primarily due to difficulty in maintaining FMNH2 in a reduced state at very low concentrations. In the present studies this difficulty has been circumvented, permitting us to elucidate the order of the bioluminescent reaction with respect to FMNH2 and the dissociation constant of the FMNH2-enzyme complex. Of particular importance was the fact that enzymatic measurements could be made not only at extremely low FMNH2 concentrations (10-9 m), but also at very high enzyme concentrations, even higher than the substrate concentration or the value for the dissociation constant for the FMNH2-enzyme complex (8 x 10-7 m). Under these conditions, the Michaelis-Menten equation is not directly applicable and the relative velocity of the reaction (v/Vmax) depends on the concentration of substrate-binding sites. Consequently, it was possible to elucidate the number of FMNH2-binding sites directly from kinetic data. These results show that bacterial luciferase possesses only a single FMNH2-binding site and consequently eliminate reaction mechanisms involving 2 FMNH2 molecules. Since the oxidation of a single FMNH2 molecule would not be adequate energetically for the emission of a quantum of light at 490 nm, additional energy-yielding steps must be required in the bacterial bioluminescent reaction.