Kinetics of inhibition of leukocyte 12-lipoxygenase by the isoform-specific inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid.

Moody JS, Marnett LJ
Biochemistry. 2002 41 (32): 10297-303

PMID: 12162745 · DOI:10.1021/bi020320q

Lipoxygenases (LOXs) are a ubiquitous family of enzymes that catalyze the dioxygenation of polyunsaturated fatty acids. Their role in a diverse range of biological processes has prompted the development of a large number of lipoxygenase inhibitors of possible therapeutic and probative value. The isoform-selective inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) was previously shown to inhibit leukocyte-type 12-LOX by a novel mechanism in which it binds to both the ferrous and ferric forms of the enzyme. The current study provides a detailed kinetic model of this inhibition. Nonlinear regression analysis of OPP's inhibition of arachidonic acid dioxygenation indicated mixed inhibition toward the ferric form of 12-LOX with apparent K(I) values in the low micromolar range: 2.0 +/- 0.2 microM for the free enzyme and 4.5 +/- 0.7 microM for the substrate-bound form of the enzyme. Rapid kinetic techniques allowed OPP's inhibition of the activation of the enzyme from the ferrous to the ferric form to be investigated. Titration of ferrous 12-LOX with OPP indicated that it bound to the ferrous form with an apparent K(I) value of 70 +/- 20 nM, suggesting a significantly higher affinity for the ferrous form than for the ferric form of the enzyme. Investigation of the LOX inhibitors nordihydroguaiaretic acid, N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea, BWA137C, and eicosatetraynoic acid revealed that eicosatetraynoic acid also inhibited the activation of 12-LOX. These results demonstrate that LOX inhibitors are capable of binding to multiple forms of LOXs with high affinity and suggest that inhibition of enzyme activation may be an unrecognized mechanism of inhibition of additional LOX inhibitors.

MeSH Terms (16)

5,8,11,14-Eicosatetraynoic Acid Animals Arachidonate 12-Lipoxygenase Binding, Competitive Binding Sites Enzyme Inhibitors Isoenzymes Kinetics Leukocytes Linoleic Acids Lipid Peroxides Lipoxygenase Inhibitors Oxidation-Reduction Oxygen Phenylpropionates Swine

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