title: 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 μM) has been measured in 50 mM Hepes buffer (pH = 7.5), 10 nM Luc, 250 μM ATP and D-luciferin (D-LH(2), 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-LH(2) 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-LH(2) were 16.1 ± 1.0, 16.6 ± 2.3 and 14.4 ± 0.96 μM, 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.
authors.affiliation: Centro de Investigação em Química (UP), Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, Porto, Portugal. jcsilva@fc.up.pt
authors.forename: Luís Pinto
authors.initials: LP
authors.lastname: da Silva
authors.forename: Joaquim C G Esteves
authors.initials: JC
authors.lastname: da Silva
mesh_terms: D000818:Animals; D003065:Coenzyme A / Q000737:chemistry*; D048888:Fireflies / Q000201:enzymology; D049454:Firefly Luciferin / Q000737:chemistry*; D007700:Kinetics; D049409:Luciferases, Firefly / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011485:Protein Binding; D013237:Stereoisomerism
chemical_list: C107793:dehydroluciferin; D049454:Firefly Luciferin; D049409:Luciferases, Firefly; D003065:Coenzyme A