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 µM 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.