A Mutagenesis Study of the Putative Luciferin Binding Site Residues of Firefly Luciferase

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate firefly luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We had previously developed [Branchini, B. R., Magyar, R. A., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry 37, 15311−15319] a molecular graphics-based working model of the luciferase active site starting with the first X-ray structure [Conti, E., Franks, N. P., and Brick, P. (1996) Structure 4, 287−298] of the enzyme without bound substrates. In our model, the luciferin binding site contains 15 residues that are within 5 A of the substrate. Using site-directed mutagenesis, we made changes at all of these residues and report here the characterization of the corresponding expressed and purified proteins. Of the 15 residues studied, 12 had a significantly (≥4-fold Km difference) altered binding affinity for luciferin and seven residues, spanning the primary sequence region Arg218−Ala348, had substantial...