Enoyl-ACP reductase (FabI) of Haemophilus influenzae: steady-state kinetic mechanism and inhibition by triclosan and hexachlorophene.
Steady-state kinetics, equilibrium binding, and primary substrate kinetic isotope effect studies revealed that the reduction of crotonyl-CoA by NADH, catalyzed by Haemophilus influenzae enoyl-ACP reductase (FabI), follows a rapid equilibrium random kinetic mechanism with negative interaction among the substrates. Two biphenyl inhibitors, triclosan and hexachlorophene, were studied in the context of the kinetic mechanism. IC(50) values for triclosan in the presence and absence of NAD(+) were 0.1 +/- 0.02 and 2.4 +/- 0.02 microM, respectively, confirming previous observations that the E-NAD(+) complex binds triclosan more tightly than the free enzyme. Preincubation of the enzyme with triclosan and NADH suggested that the E-NADH complex is the active triclosan binding species as well. These results were reinforced by measurement of binding kinetic transients. Intrinsic protein fluorescence changes induced by binding of 20 microM triclosan to E, E-NADH, E-NAD(+), and E-crotonyl-CoA occur at rates of 0.0124 +/- 0.001, 0.0663 +/- 0.002, 0.412 +/- 0.01, and 0.0069 +/- 0.0001 s(-1), respectively. The rate of binding decreased with increasing crotonyl-CoA concentrations in the E-crotonyl-CoA complex, and the extrapolated rate at zero concentration of crotonyl-CoA corresponded to the rate observed for the binding to the free enzyme. This suggests that triclosan and the acyl substrate share a common binding site. Hexachlorophene inhibition, on the other hand, was NAD(+)- and time-independent; and the calculated IC(50) value was 2.5 +/- 0.4 microM. Steady-state inhibition patterns did not allow the mode of inhibition to be unambiguously determined, but binding kinetics suggested that free enzyme, E-NAD(+), and E-crotonyl-CoA have similar affinity for hexachlorophene, since the k(obs)s were in the same range of 20-24 s(-1). When the E-NADH complex was mixed with hexachlorophene ligand, concentration-independent fluorescence quenching at 480 nm was observed, suggesting at least partial competition between NADH and hexachlorophene for the same binding site. Mutual exclusivity studies, together with the above-discussed results, indicate that triclosan and hexachlorophene bind at different sites of H. influenzae FabI.
Marcinkeviciene
J
J
Department of Chemical Enzymology, Dupont Pharmaceutical Company, Wilmington, Delaware 19880-0400, USA. jovita.marcinkeviciene@dupontpharma.com
Jiang
W
W
Kopcho
L M
LM
Locke
G
G
Luo
Y
Y
Copeland
R A
RA
D001665:Binding Sites; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D004791:Enzyme Inhibitors / Q000494:pharmacology; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics; D006582:Hexachlorophene / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014260:Triclosan / Q000494:pharmacology
D004791:Enzyme Inhibitors; D009243:NAD; D014260:Triclosan; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D006582:Hexachlorophene