Identification of a firefly luciferase active site peptide using a benzophenone-based photooxidation reagent.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a series of reactions that require MgATP and molecular oxygen. We prepared 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a novel benzophenone-based substrate analog, intending to use it in photoaffinity labeling studies to probe the luciferase active site. Instead, we found that while BPTC was a potent photoinactivating reagent for firefly luciferase, it was not a photoaffinity labeling agent. Using proteolysis, reverse phase high-performance liquid chromatography, tandem high performance liquid chromatography-electrospray ionization mass spectrometry, and Edman sequencing, we identified a single luciferase peptide, 244HHGF247, the degradation of which was directly correlated to luciferase photoinactivation. Results of enzyme kinetics and related studies were consistent with this peptide being at or near the luciferin binding site. Further, peptide model studies and additional investigations on the nature of the photoinactivation process strongly suggested that BPTC catalyzed the formation of singlet oxygen at the active site of the enzyme. We describe here an uncommon example of active site-directed photooxidation of an enzyme by singlet oxygen.

Inactivation of firefly luciferase with N-(iodoacetyl)-N'- (5-sulfo-1-naphthyl)ethylenediamine (I-AEDANS).

N-Iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (I-AEDANS), a fluorescent reagent that selectively modifies cysteine residues, was demonstrated to irreversibly inhibit native Photinus pyralis luciferase purified from firefly lanterns. Complete inactivation of luciferase activity was accompanied by the blockage of all four cysteine thiols and the concomitant incorporation of 4 mol of N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) per mole of enzyme. Employing proteolytic digestions of AEDANS-labeled luciferase and reverse-phase-high-performance liquid chromatography (RP-HPLC), seven tagged peptides were isolated. The AEDANS label provided a convenient spectroscopic marker for the identification of the modified peptides. The sequences of the labeled peptides were deduced from electrospray ionization mass spectrometry (ESMS) and N-terminal sequencing. The fluorescent peptides included cysteine residues and spanned sequences composed of amino acids Leu78-Lys85, Thr214-Arg218, Asp224-Arg275, and Gly388-Met396. The luciferin substrate provided substantial protection against luciferase inactivation resulting in a 60-67% decrease in the labeling of all four cysteine thiols. Thus, it does not appear that a specific cysteine mediates the loss of luciferase activity. Additional LC/ESMS studies permitted the identification of 78% of the native luciferase molecule, which, unlike the recombinant protein, was found to contain an acetylated N-terminus. The AEDANS labeling results and the identification of well-defined proteolytic fragments should facilitate future structure-function investigations of the firefly luciferases.