Nordihydroguaiaretic acid (NDGA), one of the most efficient inhibitors of lipoxygenases, is shown, by electron paramagnetic resonance, circular dichroism, and fluorescence studies, to reduce the catalytically active ferric soybean lipoxygenase 1 (Eox) to the inactive ferrous form (Ered). In decreasing order of reactivity, the following also reduce Eox: catechol greater than hydroquinone greater than 2,6-di-tert-butyl-4-methylphenol greater than esculetin greater than caffeic acid approximately equal to alpha-tocopherol greater than norepinephrine greater than dithiothreitol. The reduction of Eox by NDGA (kappa = 8.1 X 10(6) M-1 S-1, pH 9.0, 25 degrees C) is almost as fast as the Eox-catalyzed conversion of linoleate (LH) to 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoate (LOOH) and the oxidation of Ered by LOOH to give Eox. Thus, NDGA can efficiently inhibit the Eox-catalyzed conversion of LH to LOOH by reducing Eox to the inactive Ered, thereby diminishing the turnover rate. Lipoxygenase catalyzes the oxidation of NDGA by LOOH at a rate that is consistent with the independently determined rate constant for the reduction of Eox by NDGA. All four reducing equivalents from the two catechol groups in NDGA can be utilized in the reduction of Eox, leading to the consumption of 4 mol of LOOH/mol of NDGA initially present. Because the catalytically inactive Ered is oxidized by fatty acid hydroperoxides (e.g., LOOH) to give the active Eox, reducing agents such as NDGA are most effective as lipoxygenase inhibitors at low hydroperoxide concentrations. Our results suggest that in vivo, where lipid hydroperoxides are maintained at low steady-state levels, reduction of lipoxygenase from the ferric to ferrous state may be important in the regulation of lipoxygenase activity and hence leukotriene biosynthesis.

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The mechanism of slow binding inhibition of 3-hydroxy-3-methylglutaryl- coenzyme A reductase by lovastatin, fluindostatin, and related compounds was studied. Several of these compounds, including lovastatin, were found to be slow binding, while other less potent inhibitors were not. From a comparison of kinetic parameters obtained by steady-state measurements and progress curve analysis, it was concluded that the slow binding inhibitors bind by a mechanism which is more accurately described by biphasic binding than by single-step binding. The overall association rates of the slow binding inhibitors range from 1 x 10(6) to 4 x 10(-7) M-1 s-1, and the dissociation rates are in the range of 10(-3) s-1. The structures of slow binding and reversible inhibitors were compared by using molecular modeling methods. From these comparisons, it was proposed that the slow binding and very potent inhibition of, for instance, lovastatin, is not simply a result of binding of a transition state or reaction intermediate analogue. The various lipophilic groups of the inhibitors that do not seem to be related to structural features of the substrate may also play a crucial role in determining the mechanism of binding of HMGR inhibitors.

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