Premium
Flash properties of Gaussia luciferase are the result of covalent inhibition after a limited number of cycles
Author(s) -
Dijkema Fenne Marjolein,
Nordentoft Matilde Knapkøien,
Didriksen Anders Krøll,
Corneliussen Anders Sværke,
Willemoës Martin,
Winther Jakob R.
Publication year - 2021
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.4023
Subject(s) - luciferase , substrate (aquarium) , luciferases , chemistry , kinetics , enzyme kinetics , covalent bond , bioluminescence , biophysics , biochemistry , enzyme , biology , active site , organic chemistry , physics , quantum mechanics , ecology , transfection , gene
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.