HYDROPHOBIC NATURE OF ACTIVE SITE OF FIREFLY LUCIFERASE

Anilinonaphthalenesulfonates and toluidinonaphthalenesulfonates bind to firefly luciferase with an enhancement of fluorescence. Approximately 2 moles of dye is bound per mole of enzyme. The affinity of 2,6-toluidinonaphthalenesulfonate for the enzyme is much greater than 1,5-anilinonaphthalenesulfonate or the corresponding isomer 2,6-anilinonaphthalenesulfonate. Both 2,6-toluidinonaphthalenesulfonate and 1,5-anilinonaphthalenesulfonate are competitive inhibitors of luciferin. The addition of 2 moles of dehydroluciferyl adenylate/mole of enzyme completely removes the bound dye from the enzyme. Inhibition of the enzymatic activity by reaction of the two essential sulfhydry groups with N-ethylmaleimide decreases the affinity of the enzyme for 2,6-toluidinonaphthalenesulfonate but does not alter the number of dye molecules bound. The binding of 2,6-toluidinonaphthalenesulfonate is independent of pH between 6 and 9. The increase in fluorescent intensity and the shift in the emission maximum of the bound dye are indicative of hydrophobic binding sites on the enzyme. The data support the conclusion that the dyes bind at the normal luciferin binding sites, thus inhibiting catalytic activity. © 1969, American Chemical Society. All rights reserved.