A Novel Aromatic Carboxylic Acid Inhibits Luciferase Enzymatic Activity in Mammalian Cells by Acylation of an Active Regulatory Lysine Residue

Abstract

Firefly luciferase (Luc) is widely used as a reporter enzyme in cell-based assays for gene expression. A novel aromatic carboxylic acid, F-53 substantially inhibited the enzymatic activity of Luc in a Luc reporter screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry (MS/MS) analyses showed that F-53 modified Luc at lysine-529 via amidation of the F-53 carboxyl group. The lysine-529 residue of Luc, which plays a regulatory catalytic role, can be acetylated. Luc also has a long-chain fatty acyl-CoA synthase activity. An in vitro assay that involved both recombinant Luc and mouse liver microsomes identified F-53-CoA as the reactive form produced from F-53. However, whereas the inhibitory effect of F-53 is observed in Hela cells that transiently expressed Luc, it is not observed in an in vitro assay that involves recombinant Luc alone. Therefore, insights into the activities of certain mammalian transferases can be translated to better understand the acylation by F-53.The purification of the interacting proteins with F-53 in mammalian cells was carried out using F-53-immobilized Magnetic FG beads as bait and those were analyzed by using nano-Liquid chromatography coupled with electrospray ionization-quadrupole time-of-flight hybrid mass spectrometry (nano-LC/ESI-QTOF-MS).