Kinetics of inhibition of firefly luciferase by dehydroluciferyl-coenzyme A, dehydroluciferin and l-luciferin

Abstract

The inhibition mechanisms of the firefly luciferase (Luc) by three of the most important inhibitors of the reactions catalysed by Luc, dehydroluciferyl-coenzyme A (L-CoA), dehydroluciferin (L) and L-luciferin (L-LH₂) were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 μM) has been measured in 50 mM Hepes buffer (pH = 7.5), 10 nM Luc, 250 μM ATP and D-luciferin (D-LH₂, from 3.75 up to 120 μM). Nonlinear regression analysis with the appropriate kinetic models (Henri–Michaelis–Menten and William–Morrison equations) reveals that L-CoA is a non-competitive inhibitor of Luc (K_i = 0.88 ± 0.03 μM), L is a tight-binding uncompetitive inhibitor (K_i = 0.00490 ± 0.00009 μM) and L-LH₂ acts as a mixed-type non-competitive-uncompetitive inhibitor (K_i = 0.68 ± 0.14 μM and αK_i = 0.34 ± 0.16 μM). The K_m values obtained for L-CoA, L and L-LH₂ were 16.1 ± 1.0, 16.6 ± 2.3 and 14.4 ± 0.96 μM, respectively. L and L-LH₂ are strong inhibitors of Luc, which may indicate an important role for these compounds in Luc characteristic flash profile. L-CoA K_i supports the conclusion that CoA can stimulate the light emission reaction by provoking the formation of a weaker inhibitor.