Substrate-binding properties of firefly luciferase

Various techniques were used for characterization of the luciferin-binding site of the firefly luciferase. Both equilibrium-dialysis and fluorescence-titration techniques revealed the existence of two identical, noninteracting binding sites for dehydroluciferin, which is a potent competitive inhibitor. Kinetically obtained dissociation constants of various luciferin analogues and other competitive inhibitors revealed that most of the binding energies of these compounds reside in the backbone ring structure and that various substituents did not influence the binding to any significant extent. Exceptions to this are the methyl group at the 6-position of the benzothiazole ring and the carboxylic acid group. The binding energy of the luciferin backbone structure to the luciferin-binding site is 7.5 kcal/mole. Two parts of the luciferin molecule, the benzothiazole ring portion and the thiazoline ring portion, contribute 6.0 kcal/mole and 1.5 kcal/mole respectively to the binding energy. Comparison of the binding energies of several heterocyclic compounds structurally related to benzothiazole suggests that the nitrogen atom of the benzothiazole ring may be important in orienting the compound to a fixed position in the luciferinbinding site. The binding studies with N-ethylmaleimide-inactivated luciferase gave further evidence that the two sulfhydryl groups essential for the luciferase activity are located at the luciferin binding sites, most likely one at each binding site. Excitation spectra of luciferase-bound dehydroluciferin indicated that the phenolic group of dehydroluciferin is in unionized form on the enzyme even in a medium of high pH. This effect and a large enhancement of the blue fluorescence peak at 440 mμ suggest the environment of luciferin-binding site to be quite hydrophobic in agreement with earlier observations.