Oleyl sulfate reveals allosteric inhibition of soybean lipoxygenase-1 and human 15-lipoxygenase.
Inhibition of lipoxygenase (LO) is currently an important goal of biomedical research due to its critical role in asthma, atherosclerosis, and cancer regulation. Steady-state kinetic data indicate that oleic acid (OA) is a simple competitive inhibitor for soybean lipoxygenase; however, kinetic isotope effect (KIE) data suggest a more complicated inhibitory mechanism. To investigate the inhibitory effects of fatty acids on lipoxygenase more thoroughly, we have synthesized a novel inhibitor to lipoxygenase, (Z)-9-octadecenyl sulfate (oleyl sulfate, OS), which imparts kinetic properties that are inconsistent with simple competitive inhibition for both SLO-1 and 15-HLO. The KIE exhibits a hyperbolic rise with addition of OS, indicating the formation of a catalytically active ternary complex with K(D) values of 0.6 +/- 0.2 and 0.4 +/- 0.05 microM for SLO-1 and 15-HLO, respectively. The steady-state kinetics show that SLO-1 proceeds through a hyperbolic mixed-type inhibition pathway, where OS binding (K(i) = 0.7 +/- 0.3 microM) causes an approximate 4-fold increase in the K(m)(app) (alpha = 4.6 +/- 0.5) and a decrease in the k(cat) by approximately 15% (beta = 0.85 +/- 0.1). 15-HLO also exhibits a hyperbolic saturation of k(cat)/K(m) consistent with the observed rise in its KIE. Taken together, these findings indicate the presence of an allosteric site in both SLO-1 and 15-HLO and suggest broad implications regarding the inhibition of LO and the treatment of LO-related diseases.
Mogul
R
R
Department of Chemistry, University of California, Santa Cruz, California 95064, USA.
Johansen
E
E
Holman
T R
TR
D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site / Q000187:drug effects; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D006801:Humans; D007554:Isotopes; D007700:Kinetics; D019787:Linoleic Acid / Q000378:metabolism / Q000494:pharmacology; D008084:Lipoxygenase / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology*; D008823:Micelles; D019301:Oleic Acid / Q000378:metabolism / Q000494:pharmacology; D012995:Solubility; D013025:Glycine max / Q000201:enzymology*; D013500:Surface Tension; D013816:Thermodynamics; D014783:Viscosity
D007554:Isotopes; D016859:Lipoxygenase Inhibitors; D008823:Micelles; D019301:Oleic Acid; D019787:Linoleic Acid; D008084:Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase