IJUP08 - Universidade do Porto

Kinetics of Inhibition of Firefly Luciferase by Oxyluciferin and
Dehydroluciferyl-adenylate
César Ribeiro 1 and Joaquim C.G. Esteves da Silva 1
1 Department of Chemistry, CIQ-UP, Faculty of Sciences, University of Porto, Portugal.
Firefly luciferase (Luc) catalyzes the oxidation of D-luciferin (LH2) with molecular
oxygen in the presence of ATP and Mg 2+ with emission of yellow-green light in an
extremely efficient process [1]. This reaction involves the formation of an enzyme bound
adenylyl intermediate (LH2-AMP) (Eq. 1) following oxidation with release of AMP,
pyrophosphate, CO2 and oxyluciferin (Eq. 2), the assumed light emitter and also an
inhibitor of luciferase. Dehydroluciferyl-adenylate (L-AMP), a potent inhibitor of light
emission, is also formed as a side product of the bioluminescence reaction.
Luc + LH2 + ATP → Luc·LH2-AMP + pyrophosphate (1)
Luc·LH2-AMP + O2 → Luc + AMP + CO2 + oxyluciferin + photon (2)
The light emission starts with an initial flash that quickly decays to a low basal level due to
accumulation of inhibitory products. So far no conclusive explanation has been proposed
regarding the kinetic mechanism of inhibition and the relative importance of the inhibitory
products.
The applications of luciferase include a remarkable number of biochemical and clinical
tools, many of which use the luciferase gene as a reporter of gene expression and
regulation. Luciferase is also a very sensitive analytical tool for detection of ATP.
The current study provides a detailed kinetic model of the inhibition exerted by
oxyluciferin and L-AMP by measuring the light production in the presence and absence of
these inhibitors (Fig. 1). Nonlinear regression analysis suggest that oxyluciferin is a
competitive inhibitor of luciferase (Ki = 1 – 3 μM) while L-AMP act as a tight-binding
competitive inhibitor (Ki = 2 nM).
RLU
1500000
1000000
500000
0
0.0 0.5 1.0 1.5 2.0 2.5
t/s
0 uM Oxyluciferin
2.5 uM Oxyluciferin
5.0 uM Oxyluciferin
RLU
200000
150000
100000
50000
0
0.0 1.0 2.0
t/s
3.0 4.0
0 nM L-AMP
40 nM L-AMP
60 nM L-AMP
Fig.1. Inhibitory effect of oxyluciferin and L-AMP on firefly luciferase bioluminescence. Mixtures
cointaining ATP 50 μM and LH2 10 μM were injected into solutions of Luc. 10 nM in hepes buffer
(pH=7.5) pre-incubated with inhibitor.
References:
[1] McElroy, W.D., Seliger, H.H. and White, E.H. (1969), Mechanism of Bioluminescence,
Chemiluminescence and Enzyme Function in the Oxidation of Firefly Luciferin, Photochemistry
and Photobiology, 10 (3), 153-170.
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