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.
Branchini
B R
BR
Central Research Division, Pfizer Inc., Groton, Connecticut 06340, USA. brbra@conncol.edu
Magyar
R A
RA
Marcantonio
K M
KM
Newberry
K J
KJ
Stroh
J G
JG
Hinz
L K
LK
Murtiashaw
M H
MH
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D001517:Coleoptera; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010782:Photolysis
D000345:Affinity Labels; D008156:Luciferases