IJUP08 - Universidade do Porto

Effect of pyrophosphate on firefly luciferase bioluminescence
Filipe Peralta 1 , Joaquim C.G. Esteves da Silva 1 and Rui Fontes 2
1 Department of Chemistry, CIQ-UP, Faculty of Sciences, University of Porto, Portugal.
2 Serviço de Bioquímica (U38-FCT), Faculty of Medicine, University of Porto, Portugal.
Firefly luciferase (LUC, EC 1.13.12.7) is an enzyme that catalyses the oxidation of firefly
luciferin (LH2) giving rise to light [1]. The bioluminescence reaction involves the
formation, from LH2 and ATP, of an enzyme-bound adenylyl intermediate (LUC•LH2-
AMP) and its subsequent oxidation with release of AMP, CO2 and oxyluciferin in an
electronically excited state.
LUC + LH2 + ATP ⇌ LUC•LH2-AMP + PPi (1)
LUC•LH2-AMP + O2 → LUC + AMP + CO2 + oxyluciferin + photon (2)
Inorganic pyrophosphate (PPi) was described as stabilizer and/or activator of firefly
bioluminescence [2]. However, PPi effect is both intriguing and paradoxical: since PPi is a
product of the adenylation step (reaction 1), an inhibitory effect would be expected and can
indeed be observed but, as stated, PPi can also act as a stabilizer or activator of LUC
bioluminescence.
In this communication we show that the stabilizing and activating effect of PPi on LUC
bioluminescence can be explained by the theory proposed by McElroy et al [2]. This
theory proposes the pyrophosphorolysis of the enzyme-formed inhibitor dehydroluciferyladenylate
(L-AMP) giving rise to dehydroluciferin (L) and ATP (Eq. 3) - L is a much less
powerful inhibitor than L-AMP.
LUC•L-AMP + PPi ⇌ LUC + L + ATP (3)
Consequently, this reaction converts L-AMP into L allowing the removal of a powerful
inhibitor (L-AMP) from the enzyme active site and its replacement by a less powerful
inhibitor (L).
Acknowledgements: Financial support from Universidade do Porto and Caixa Geral de
Depósitos (Project IPG136) is acknowledged.
References:
[1] Nakatsu, T., Ichiyama, S., Hiratake, J., Saldanha, A., Kobashi, N., Sakata, K., and
Kato, H. (2006) Structural basis for the spectral difference in luciferase bioluminescence.
Nature 440, 372-376
[2] Rhodes, W. C., and McElroy, W. D. (1958) The synthesis and function of luciferyl-adenylate
and oxyluciferyl-adenylate. J Biol Chem 233, 1528-1537.
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