Flash properties of Gaussia luciferase are the result of covalent inhibition after a limited number of cycles

Abstract

Luciferases are widely used as reporters for gene expression and for sensitive detection systems. The luciferase (GLuc) from the marine copepod Gaussia princeps, has gained popularity, primarily because it is secreted and displays a very high light intensity. While firefly luciferase is characterized by kinetic behavior which is consistent with conventional steady‐state Michaelis–Menten kinetics, GLuc displays what has been termed “flash” kinetics, which signify a burst in light emission followed by a rapid decay. As the mechanistic background for this behavior was unclear, we decided to decipher this in more detail. We show that decay in light signal is not due to depletion of substrate, but rather is caused by the irreversible inactivation of the enzyme. Inactivation takes place after between 10 and 200 reaction cycles, depending on substrate concentration and can be described by the sum of two exponentials with associated rate constants. The dominant of these increases linearly with substrate concentration while the minor is substrate‐concentration independent. In terms of rate of initial luminescence reaction, this increases with the substrate concentration to the power of 1.5 and shows no signs of saturation up to 10 μM coelenterazine. Finally, we find that the inactivated form of the enzyme has a larger apparent size in both size exclusion chromatography and SDS‐PAGE analysis and shows a fluorescence peak at 410 nm when excited at 333 nm. These findings indicate that the “flash” kinetics in Gaussia luciferase are caused by an irreversible covalent binding to a substrate derivative during catalysis.

A protein aggregation platform that distinguishes oligomers from amyloid fibrils

“…The flash properties of Gaussia luciferase has been shown to be caused by covalent inactivation of the enzyme. 60 Because luciferase is not related to cell survival, detecting changes in Aβ aggregation can only be achieved through bioluminescence measurements, limiting the use of the split Gaussia luciferase system in high-throughput screening.…”

Microliter Scale Synthesis of Luciferase‐Encapsulated Polymersomes as Artificial Organelles for Optogenetic Modulation of Cardiomyocyte Beating

“…Some loss of GLuc activity was observed after each stimulation which has previously been observed when free GLuc is subjected to repeated CTZ stimulation and has been attributed to an irreversible covalent binding event that occurs during the enzymatic transformation. [26] To demonstrate the potential of GLuc/PSomes as AOs, we first confirmed their biocompatibility with iPSC-CMs (>85% purity based on flow cytometry of cardiac troponin T (cTnT) staining, Figure S9, Supporting Information) by incubating cells with empty PSomes or GLuc/PSomes for 3 days and analyzing cell viability with the PrestoBlue metabolic assay (Figure S10, Supporting Information). Under these conditions, both empty and GLucloaded PSomes were not cytotoxic up to polymer concentrations of 1600 μg mL -1 (corresponding to a maximum GLuc concentration of 800 ng mL -1 ) which is in line with literature reports of negligible cytotoxicity of PEG-b-PHPMA nanoparticles.…”

“…Some loss of GLuc activity was observed after each stimulation which has previously been observed when free GLuc is subjected to repeated CTZ stimulation and has been attributed to an irreversible covalent binding event that occurs during the enzymatic transformation. [ 26 ]…”

Miniaturization of Bright Light-Emitting Luciferase ALuc: picALuc

“…33 Thus far, one report has described an inhibitor for copepod luciferase. 34 However, the inhibitor has not been identified. Identification of this inhibitor and the discovery of a suppressor against this inhibitory effect are required for more accurate bioluminescence measurements of copepod luciferases, including picALuc.…”

“…In this reaction, the inhibitor dehydroluciferyl-adenylate (L-AMP) is formed as a byproduct; moreover, the addition of coenzyme A can remarkably reduce the inhibitory effect of L-AMP . Thus far, one report has described an inhibitor for copepod luciferase . However, the inhibitor has not been identified.…”