A kinetic model for lipoxygenases based on experimental data with the lipoxygenase of reticulocytes

Abstract

A comprehensive kinetic model for lipoxygenase catalysis is proposed which includes the simultaneous occurrence of dioxygenase and hydroperoxidase activities and is based on the assumption of a single binding site for substrate fatty acid and product.The aerobic reaction of purified lipoxygenase from rabbit reticulocytes with 9,12(Z,Z)-octadecadienoic acid (linoleic acid) as substrate was studied.The rate constants and the dissociation constants of this enzyme were calculated for the model from progress curves; the model describes correctly the experimental data.The following kinetic features of the reticulocyte enzyme are assumed to apply generally to lipoxygenases. As predicted from the model it was found that at low concentrations of oxygen the regio-and stereospecificities of the dioxygenation are diminished.During the autoactivation phase the steady-state approximation does not hold.Animal lipoxygenases (EC 1.1 3.1 1.12) have acquired particular interest owing to their key role in the biosynthesis of leukotrienes and other biologically active products of their action. So far research on their actions has been focussed on their positional and stereospecificity [l] ; mechanistic studies on their kinetics have hardly been performed. The situation is different with respect to plant enzymes, in particular soybean lipoxygenase, the kinetics of which has been studied extensively [2 -61. We undertook to set up a viable kinetic model for the reticulocyte lipoxygenase which may be considered to be a prototype of the animal lipoxygenases; this enzyme is available in purified form, sufficient amounts and has been the object of some mechanistic work by us [7]. In view of the many properties shared by plant and animal lipoxygenases we scrutinized the models proposed so far for the soybean enzyme as to their applicability to the reticulocyte enzyme and found them to be generally unsatisfactory in two respects.a is difficult to reconcile with the successful and well-tested onesite model for the anaerobic lipohydroperoxidase activity of soybean lipoxygenase [6]. A transition from a one-site catalysis under anaerobic conditions to a two-site catalysis under aerobic ones would imply either the existence of different active sites for hydroperoxidase and dioxygenase activity or the unmasking or forming of a second site by dioxygen.Therefore we tried to elaborate a one-site model which will describe both dioxygenase and concomitant hydroperoxidase activities of lipoxygenase using the kinetic model of Verhagen et al. [6] as starting point. This model and some of its predictions were tested on the reticulocyte lipoxygenase with 9,12(Z,Z)-octadecadienoic acid (linoleic acid) as substrate.The model elaborated is assumed to be applicable to study the kinetic behaviour of other lipoxygenases. MATERIALS AND METHODSLipoxygenase from reticulocytes was isolated and purified as described elsewhere [lo]. A peak fraction of the isoelectricfocussed enzyme was used with a specific activity of 8 s-and a protein content of 3.05mg/ml. Differen...

A Kinetic Model for the Interaction of Nitric Oxide with a Mammalian Lipoxygenase

“…In a previous report [24] we introduced a kinetic model for the rabbit 15-lipoxygenase-catalysed oxidation of linoleic acid which has been also adapted for the soybean enzyme [17]. This model, which is depicted in a modified form in Scheme 1, consists mainly of three parts: (a) Activation of the native enzyme by the reaction product 13-hydro- Irreversible auto-inactivation of the lipoxygenase.…”

“…The progress curves of rabbit 15-lipoxygenase-catalysed oxygenation of polyenoic fatty acids can be divided into three parts [24]: (a) a kinetic lag phase, (b) a linear progression phase and (c) an exhaustion phase which is mainly due to irreversible inactivation of the enzyme. The kinetic lag phase can be abolished when the reaction product (9Z, 1 1 0 -13-hydroperoxy-9,ll-octadecadienoic acid is added, whereas the irreversible enzyme inactivation is slowed down if the reaction is performed at 24°C [24].…”

“…Numerical values of those kinetic parameters which are not linked to the action of NO were taken from [24].…”

Effect of lipid hydroperoxide on lipoxygenase kinetics

“…Studies on the isotope effect in the anaerobic reaction (Verhagen, 1978) provided further evidence that the proposed mechanism and the kinetic constants calculated on the basis of this model are correct. The model for the anaerobic reaction has been elaborated by Ludwig et al (1987) to include the aerobic reaction. Many parameter values reported for reticulocyte lipoxygenase in Ludwig et al (1987) are, however, very different from the corresponding values in the anaerobic soybean lipoxygenase-1 reaction.…”

The Dioxygenation Rate In Lipoxygenase Catalysis Is Determined by the Amount of Iron(III) Lipoxygenase in Solution

“…In the two-step model (De Groot et al, 1975b;Ludwig et al, 1987;Schilstra et al, 1992), only iron(II1) lipoxygenase can catalyze the first step of the reaction, namely, hydrogen abstraction from S to form the radical Sox. In the second reaction step, insertion of 0 2 at C-1 or C-5 of the pentadiene system leads to the formation of P. Hydrogen abstraction is coupled to reduction of the iron cofactor to Fe(II), and 0 2 insertion is accompanied by its reoxidation to Fe(II1).…”