Firefly luciferase catalyzes series of reactions to efficiently yield its bioluminescence using two substrates luciferin and ATP. This reaction process can be largely divided into two half reactions: adenylation of luciferin and oxidative reactions using luciferyl adenylate (LH2-AMP). The luciferase, as a member of acyl-adenylate enzyme superfamily, is composed of two (N-terminal and C-terminal) domains as well as other enzymes, and the two domains has been thought to cooperate together for the light-emitting reactions.

We found that although weak (and slow), the N-terminal domain alone has its luminescence activity using the two substrates, and also found that we can specifically and sensitively detect the reaction intermediate LH2-AMP in the presence of excess luciferin and ATP by using this domain based on the different kinetics. This enabled us to measure the LH2-AMP concentration during the reactions of various mutants, which is expected to lead to the elucidation of the not fully understood reaction mechanism. Also expected is the development of protein-protein interaction detection method based on the mutant enzymes (FlimPIA).

References

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3) Y. Ohmuro-Matsuyama, et al. (2013) Anal. Chem. 85, 7935-7940.[DOI][Abstract]
4) Press release by JST (2013)
5) Y. Ohmuro-Matsuyama, et al. (2014) Anal. Chem. 86, 2013-2018. [DOI][Abstract]

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