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-LH(2)) were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 mu M) has been measured in 50 mM Hepes buffer (pH = 7.5), 10 nM Luc, 250 mu M ATP and D-luciferin (D-LH(2), from 3.75 up to 120 mu M). 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 mu M), L is a tight-binding uncompetitive inhibitor (K(i) = 0.00490 +/- 0.00009 mM) and L-LH2 acts as a mixed-type non-competitive-uncompetitive inhibitor (K(i) = 0.68 +/- 0.14 mMand alpha K(i) = 0.34 +/- 0.16 mu M). The Km values obtained for L-CoA, L and L-LH(2) were 16.1 +/- 1.0, 16.6 +/- 2.3 and 14.4 +/- 0.96 mu M, respectively. L and L-LH(2) 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.