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

Luciferases are widely used as reporters for gene expression and for sensitive detection systems. While luciferases from firefly and Renilla have long been used for analysis of intracellular expression, the luciferase (GLuc) from the marine copepod Gaussia princeps, has gained popularity, primarily because it is secreted and displays a very high light intensity. Firefly luciferase is characterized by kinetic behavior which is consistent with conventional steady-state Michaelis-Menten kinetics (termed "glow" kinetics). GLuc, conversely, 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 is poorly characterized, we decided to decipher the mechanism 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. We found that the rate of inactivation is described by the sum of two exponentials with associated rate constants. The dominant of these 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 1.53 and shows no signs of saturation up to 10 M coelenterazine. Finally, we found 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 derivative of the substrate during the reaction.