title: A kinetic model for the interaction of nitric oxide with a mammalian lipoxygenase.
abstract: Nitric oxide (NO) has been known for a long while to act as an inactivator of the soybean lipoxygenase-1 and cyclooxygenase. More recently, NO was shown to interact also with a mammalian 15-lipoxygenase [Wiesner, R., Rathmann, J., Holzhütter, H. G., Stosser, R., Mader, K., Nolting, H. & Kühn, H. (1996) Nitric oxide oxidises ferrous mammalian lipoxygenases to a pre-activated ferric species, FEBS Lett. 389, 229-232]. In this paper we present a detailed kinetic analysis of the interaction of NO with the 15-lipoxygenase from reticulocytes. Time courses of hydroperoxide formation were monitored after an anaerobic incubation of the enzyme with varying concentrations of NO and for varying length of the incubation period. Owing to the presence of O2 during the enzymatic reaction, the inhibitory effect of NO declines in a time-dependent manner since NO is converted into non-reactive NO2. This is manifested as a lag phase of the time course. The lag phase becomes more pronounced if the enzyme is incubated over a fixed period (15 s) with increasing concentrations of NO. In contrast, increasing the length of the incubation period at fixed concentrations of NO diminishes the duration of the lag phase. These experimental findings can be accounted for by a kinetic model which assumes (a) fast reversible binding of NO to the inactive ferrous Fe(II) form of the enzyme and (b) slow irreversible conversion of the Fe(II)-NO complex into an activated ferric form of the enzyme which is susceptible to peroxide activation. The rate constants for these two different kinetic modes of NO action were estimated by fitting the solutions of the model equations to the experimental time courses. The dissociation constant of the Fe(II)-NO complex amounts to 2.5 microM. Computer simulations performed on the basis of the model indicate that the response of the lipoxygenase to increasing intracellular NO concentrations depend upon the available peroxide tone: at low peroxide concentration the enzyme is initially trapped in the inactive ferrous form and thus may be activated by NO via the activated ferric species. On the other hand, at high peroxide concentrations, a partial inhibition of the initially active enzyme is expected.
authors.affiliation: Institute of Biochemistry, University Clinics Charité, Humboldt University, Berlin, Germany.
authors.forename: H G
authors.initials: HG
authors.lastname: Holzhütter
authors.forename: R
authors.initials: R
authors.lastname: Wiesner
authors.forename: J
authors.initials: J
authors.lastname: Rathmann
authors.forename: R
authors.initials: R
authors.lastname: Stösser
authors.forename: H
authors.initials: H
authors.lastname: Kühn
mesh_terms: D000818:Animals; D006801:Humans; D007700:Kinetics; D008084:Lipoxygenase / Q000378:metabolism*; D008322:Mammals; D008956:Models, Chemical; D008962:Models, Theoretical; D009569:Nitric Oxide / Q000378:metabolism*
chemical_list: D009569:Nitric Oxide; D008084:Lipoxygenase