Study on the initial kinetics of yeast phosphofructokinase by stopped-flow measurements

The initial kinetics of yeast phosphofructokinase was studied by stopped-flow measurements over an enzyme concentration range from 0.5 mg/ml to 0.01 mg/ml. Before attaining the steady state the reaction showed a lag phase in the product formation, the duration of which was found to decrease with increasing enzyme concentration. The lag phase disappeared after preincubation of the enzyme for at least five minutes with either fructose 6-phosphate, fructose 1,6-bisphosphate or fructose 2,6-bisphosphate. Preincubation of the enzyme with either AMP or ADP resulted in a reduction of this phase, while ATP was without effect. Simultaneous addition of fructose 1,6-bisphosphate to the reaction mixture of the enzyme causes a significant shortening of the transient phase, whereas micromolar concentrations of fructose 2,6-bisphosphate are capable of abolishing the lag phase completely. The occurrence of an initial transient phase suggests that the enzyme after starting the reaction converts from a state of low activity to one of high activity. This conversion mainly depends on the concentration of fructose 1,6-bisphosphate generated in the course of the reaction. In addition an association reaction of the enzyme seems to be involved in the process of conversion of the phosphofructokinase during the initial transient phase.     

An autocatalytic step in the reaction cycle of hydrogenase from Thiocapsa roseopersicina can explain the special characteristics of the enzyme reaction

A moving front has been observed as a special pattern during the hydrogenase-catalyzed reaction of hydrogen uptake with benzyl viologen as electron acceptor in a thin-layer reaction chamber. Such fronts start spontaneously and at random times at different points of the reaction chamber; blue spheres are seen expanding at constant speed and amplitude. The number of observable starting points depends on the hydrogenase concentration. Fronts can be initiated by injecting either a small amount of completed reaction mixture or activated hydrogenase, but not by injecting a low concentration of reduced benzyl viologen. These characteristics are consistent with an autocatalytic reaction step in the enzyme reaction. The special characteristics of the hydrogen-uptake reaction in the bulk reaction (a long lag phase, and the enzyme concentration dependence of the lag phase) support the autocatalytic nature. We conclude that there is at least one autocatalytic reaction step in the hydrogenase-catalyzed reaction. The two possible autocatalytic schemes for hydrogenase are prion-type autocatalysis, in which two enzyme forms interact, and product-activation autocatalysis, where a reduced electron acceptor and an inactive enzyme form interact. The experimental results strongly support the occurrence of prion-type autocatalysis.     

Kinetics of activation of L-lactate dehydrogenase from Streptococcus lactis by fructose 1,6-bisphosphate

A lag is observed before the steady state during pyruvate reduction catalysed by lactate dehydrogenase from Streptococcus lactis. The lag is abolished by preincubation of enzyme with the activator fructose 1,6-bisphosphate before mixing with the substrates. The rate constants for the lag phase showed a linear dependence on fructose-1,6-bisphosphate concentration, with a second-order rate constant of 2.0 X 10(4) M-1 s-1, but were independent of enzyme concentration. Binding of fructose 1,6-bisphosphate produces a decrease in the protein fluorescence of the enzyme. The second-order rate constant for the fluorescence change is twice that for the lag in pyruvate reduction. The results suggest that binding of fructose 1,6-bisphosphate induces a conformational change in the enzyme, producing a form with reduced protein fluorescence and increased activity towards pyruvate reduction.     

Hydroxylating activity of tyrosinase and its dependence on hydrogen peroxide

The aim of this work was to study the hydroxylation of N, N-dimethyltyramine (DMTA) by tyrosinase in the presence of hydrogen peroxide, a reaction that does not take place without the addition of the hydrogen peroxide. Some properties of this hydroxylating activity are analyzed. The kinetic parameters of mushroom tyrosinase toward hydrogen peroxide (K(m) = 0.5 mM, V(m) = 11 microM/min, V(m)/K(m) = 2.2 x 10(-2) min(-1)) and toward DMTA (K(m) = 0.3 mM, V(m) = 4.8 microM/min, V(m)/K(m) = 16 x 10(-2) min(-1)) were evaluated. There was a lag period, which was similar to the characteristic lag of monophenolase activity at the expense of molecular oxygen. The length of this lag phase decreased with increasing hydrogen peroxide concentration, and disappeared at approximately 0.5 mM H(2)O(2). However, the lag was longer with higher DMTA concentrations. The pH optimum range for this hydroxylating activity was 6.0 to 7.0. The lag also varied with pH, increasing at pH values higher than 6.7. The presence of hydrogen peroxide is necessary for the oxidation of DMTA, as is the presence of active enzyme since the reaction was completely inhibited when selective tyrosinase inhibitors were added.     

Hysteresis and positive cooperativity of iceberg lettuce polyphenol oxidase.

A kinetic study of the diphenolase activity of latent polyphenol oxidase (PPO), purified from Iceberg lettuce (Lactuca sativa L), revealed a sigmoid relationship between the reaction rate and the substrate concentration with a high Hill coefficient (n(H) = 3.8). This positive cooperativity had not been previously described for any PPO. Furthermore, the enzyme showed a lag phase in the expression of this activity, suggesting a hysteretic nature of the enzyme. The kinetic behavior, the latency and the lag phase varied at different steps of the purification process. PPO showed hyperbolic or cooperative kinetics depending on the pH assay and the sodium dodecyl sulfate (SDS) concentration. Substrate-induced slow conformational change of the oligomeric enzyme is suggested. The conformational change would be toward a more active enzyme form with higher affinity for the substrate and favoured by acid pH and SDS.     

Kinetics of activation of L-lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate and by metal ions

The lactate dehydrogenase from Streptococcus faecalis is activated either by fructose 1,6-bisphosphate or by divalent cations such as Mn2+ or Co2+. With both types of activator, a lag is observed before attainment of the steady state rate of pyruvate reduction if the activator is added to the enzyme at the same time as the substrates. This lag can be largely abolished by preincubation of enzyme with activator before mixing with substrates. For fructose 1,6-bisphosphate (Fru(1,6)P2) as the activator, the rate constant for the lag phase showed a linear dependence on activator concentration but was independent of enzyme concentration. This suggests that binding of fructose 1,6-bisphosphate induces a conformational change in the enzyme which leads to increased activity, without association of enzyme subunits or dimers. With Co2+ as activator, the rate constant for the lag phase showed a hyperbolic dependence on Co2+ concentration and was also dependent on enzyme concentration. This suggests that activation by Co2+, in contrast to that by Fru(1,6)P2, involves association of enzyme dimers, followed by ligand binding.     

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

The mechanism of inhibition of porcine leukocyte 12-lipoxygenase by 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) was investigated. This compound is selective for the leukocyte form of the 12-lipoxygenase and inhibits the purified recombinant enzyme with an IC(50) value of approximately 2 microM. OPP induced a concentration-dependent lag phase in the oxygenation of arachidonic acid and decreased the maximal rate of reaction. Addition of the fatty acid hydroperoxide 13(S)-hydroperoxyoctadecadienoic acid (13-HPODE) to the reaction greatly reduced the OPP-induced lag. Lineweaver-Burk analysis of the effect of OPP on 12-lipoxygenase kinetics with arachidonic acid indicated that it was a mixed-type inhibitor. OPP was not metabolized by 12-lipoxygenase as evidenced by its quantitative recovery from incubations with stoichiometric amounts of enzyme and 13-HPODE or arachidonic acid. OPP inhibited the pseudoperoxidase activity of the enzyme with 13-HPODE and the reducing agent, BWA137C. Lineweaver-Burk analysis of the effect of OPP on pseudoperoxidase kinetics suggested that OPP was competitive with 13-HPODE. Single-turnover experiments indicated that OPP inhibited the reduction of 13-HPODE by a stoichiometric amount of ferrous 12-lipoxygenase. Addition of 13-HPODE shortened the OPP-induced lag phase but did not affect the maximal rate of enzyme activity. In addition, OPP had no effect on total product formation in either the presence or the absence of 5 microM 13-HPODE when the reaction was allowed to go to completion. All of these observations are consistent with a model for inhibition of 12-lipoxygenase activity in which OPP slows the oxidation of the inactive ferrous enzyme to the active ferric enzyme and competes with arachidonic acid for the ferric enzyme.     

Characterization of the oxidized states of bromoperoxidase

Bromoperoxidase Compound I has been formed in reactions between bromoperoxidase and organic peroxide substrates. The absorbance spectrum of bromoperoxidase Compound I closely resembles the Compound I spectra of other peroxidases. The pH dependence of the second order rate constant for the formation of Compound I with hydrogen peroxide demonstrates the presence of an ionizable group at the enzyme active site having a pKa of 5.3. Protonation of this acidic group inhibits the rate of Compound I formation. This pKa value is higher than that determined for other peroxidases but the overall pH rate profiles for Compound I formation are similar. The one-electron reduction of bromoperoxidase Compound I yields Compound II and a second reduction yields native enzyme. Bromoperoxidase Compound II readily forms Compound III in the presence of an excess of hydrogen peroxide. Compound III passes through an as yet uncharacterized intermediate (III) in its decay to native enzyme. Compound III is produced and accumulates in enzymatic bromination reactions to become the predominate steady state form of the enzyme. Since Compound III is inactive as catalyst for enzymatic bromination, its accumulation leads to an idling reaction pathway which displays an unusual kinetic pattern for the bromination of monochlorodimedone.     

The reaction between indole 3-acetic acid and horseradish peroxidase

Three distinct phases of the reaction between indole 3-acetic acid (IAA) and horse-radish peroxidase (isoenzymes B and C) were observed. When 100 μm IAA was added to an aerobic solution of the 7μm enzyme at pH 5.0 the oxidation of IAA occurred after a lag time of several seconds, during which the enzyme was partially converted into peroxide Compound II. At a time when the lag time was over the conversion of the enzyme into a green hemoprotein, called P-670 suddenly occurred at a considerable speed. The oxidation of IAA was almost over at the end of the second phase. The last phase was the restoration of the free enzyme from the remaining Compound II.Ascorbate and cytochrome c peroxidase elongated the lag phase of IAA oxidation. From these inhibition experiments it was suggested that a peroxide form of IAA would react with peroxidase to form its peroxide compounds as does hydrogen peroxide and cause the oxidation of IAA. A reaction path that the enzyme is directly reduced by IAA might be involved as an initiation step but appeared to play no essential role in the oxidation of IAA at steady state.Contrary to the cases with dihydroxyfumarate and NADH, Superoxide dismutase did not inhibit the aerobic oxidation of IAA by peroxidase. IAA peroxide radical instead of superoxide anion radical was suggested to be an intermediate in the oxidation of IAA.On the basis of stoichiometric relation of reactions between IAA and peroxidase peroxide compounds a tentative scheme of P-670 formation during the oxidation of IAA was presented.     

Catalytic pathways of Euphorbia characias peroxidase reacting with hydrogen peroxide

The reaction of Euphorbia characias latex peroxidase (ELP) with hydrogen peroxide as the sole substrate was studied by conventional and stopped-flow spectrophotometry. The reaction mechanism occurs via three distinct pathways. In the first (pathway I), ELP shows catalase-like activity: H2O2 oxidizes the native enzyme to compound I and subsequently acts as a reducing substrate, again converting compound I to the resting ferric enzyme. In the presence of an excess of hydrogen peroxide, compound I is still formed and further reacts in two other pathways. In pathway II, compound I initiates a series of cyclic reactions leading to the formation of compound II and compound III, and then returns to the native resting state. In pathway III, the enzyme is inactivated and compound I is converted into a bleached inactive species; this reaction proceeds faster in samples illuminated with bright white light, demonstrating that at least one of the intermediates is photosensitive. Calcium ions decrease the rate of pathway I and accelerate the rate of pathways II and III. Moreover, in the presence of calcium the inactive stable verdohemochrome P670 species accumulates. Thus, Ca2+ ions seem to be the key for all catalytic pathways of Euphorbia peroxidase.     

Hysteretic enzyme response induced by inhibitory antibodies against human leukocyte elastase

Polyclonal antibodies raised in rabbits against human leukocyte elastase contained two distinct populations of enzyme-inhibiting immunoglobulins. The enzyme-catalyzed reaction in the presence of antibodies (both IgG or monovalent Fab fragments) showed a transient state lasting up to several minutes depending on the inhibitor and substrate concentrations, which was followed by a linear steady-state. The transient was a concave upward or concave downward lag phase depending on whether the enzyme had been preincubated with the antibodies or not, respectively. The kinetic analysis of reaction progress curves showed that both antibody populations were slow inhibitors, which completely and reversibly excluded the substrate from binding to the enzyme. For both antibody populations, the formation of the enzyme-inhibitor complex was characterized by an initial rapid interaction followed by a slow isomerization to a catalytically inactive complex. The apparent pseudo first-order rate constant of the transient slow phase was a hyperbolic function of the inhibitor concentration for both antibodies, from which relevant kinetic constants and the half times for enzyme inactivation could be calculated. For instance, with a total antibody concentration of 1 mg/ml (as IgG), leukocyte elastase was inactivated with t1/2 = 0.31s and 24.8s by the faster and the slower of the two antibodies, respectively. It is suggested that the hysteretic response of the enzyme to the inhibitory action of its antibodies may be due to a kind of memory of the antibody molecule for a special inactive enzyme conformation resulting from inhibition by proteinase inhibitors during the immunization procedure. In turn, the purified antibodies would be able to reversibly induce a slow transition of the enzyme molecule from an active to a substrate-excluding conformation ("induced misfit").     

Fast kinetics analysis of the peroxidatic reaction catalysed by horse liver alcohol dehydrogenase

The kinetics of the enzymatic step of the peroxidatic reaction between NAD and hydrogen peroxide, catalysed by horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), has been investigated at pH 7 at high enzyme concentration. Under such conditions no burst phase has been observed, thus indicating that the rate-limiting step in the process, which converts NAD into Compound I, either precedes or coincides with the chemical step responsible for the observed spectroscopic change. Kinetic analysis of the data, performed according to a simplified reaction scheme suggests that the rate-limiting step is coincident with the spectroscopic (i.e., chemical) step itself. Furthermore, the absence of a proton burst phase indicates the proton release step does not precede the chemical step, in contrast with the case of ethanol oxidation. A kinetic effect of different premixing conditions on the reaction rate has been observed and attributed to the presence of NADH formed in the 'blank reaction' between NAD and residual ethanol tightly bound to alcohol dehydrogenase. A molecular mechanism for the enzymatic peroxidation step is finally proposed, exploiting the knowledge of the much better known reaction of ethanol oxidation. Inhibition of this reaction by NADH has been investigated with respect to H2O2 (noncompetitive, Ki about 10 microM) and to NAD (competitive, Ki about 0.7 microM). The effect of temperature on the steady-state reaction state (about 65 kJ/mol activation energy) has also been studied.     

Substrate-induced structural changes of the pyruvate dehydrogenase multienzyme complex

The time course of the overall reaction catalyzed by the pyruvate dehydrogenase multienzyme complex produces an unexpectedly high lag (tau = 8 S) even in the presence of saturating concentrations of its substrates. The preincubation of the pyruvate dehydrogenase complex with one of the substrates alone decreases the duration of this lag, and all the substrates of the pyruvate dehydrogenase component (E1) and dihydrolipoyl transacetylase component (E2) together (pyruvate, thiamine pyrophosphate, and CoA) result in the complete disappearance of the lag. The reduction of the dihydrolipoyl dehydrogenase component (E3) of the pyruvate dehydrogenase complex with the substrates of the complex in the absence of NAD+ produces significantly different quenching in the FAD fluorescence, and then the reduction with the substrates of E3 as dihydrolipoic acid and dithioerythritol. (The formation of FADH2 was not observed in the system.) The higher fluorescence quenching in the presence of substrates of pyruvate dehydrogenase complex compared to the effect caused by the substrates of the E3 component (dihydrolipoic acid and DTE) indicates conformational changes additionally manifested in the fluorescence properties of the enzyme complex. The substrate-induced quenching of the enzyme-bound FAD fluorescence shows biphasic kinetics. The rate constant of the slow phase is comparable with the rate constant calculated from the time duration of the lag phase observed in the overall reaction. The kinetic analysis of both intensity and anisotropy decrease of the FAD fluorescence suggests a consecutive transmittance of an all substrate-coordinated, induced conformational changes directed from the pyruvate dehydrogenase-via the lipoyl transacetylase--to the lipoyl dehydrogenase. Two simultaneous conformational effects caused by binding of the substrates can be distinguished; one of them results the fluorescence of the bound FAD to be more quenched, while the other makes the FAD more mobile. The first-order rate constants of both these conformational changes were determined. The present observations suggest that the pyruvate dehydrogenase complex exists in a partially inactive state in the absence of its substrates, and it becomes active due to conformational changes caused by the binding of its substrates.     

Regulatory properties of the pyruvate dehydrogenase complex from Escherichia coli. Studies on the thiamin diphosphate-dependent lag phase

The pyruvate dehydrogenase complex from Escherichia coli shows an appreciable lag phase (tau) of some minutes when its overall reaction rate was tested with very limiting amounts of thiamin diphosphate. tau depends on the concentration of thiamin diphosphate in a nonlinear fashion. Sodium diphosphate, a competitive inhibitor with respect to thiamin diphosphate (Ki = 5.2 . 10(-4) M) prolongs the lag, while the strongly binding transition state analog thiamin thiazolone diphosphate has no effect. tau is independent of the enzyme concentration, thus no dissociation-association step is involved. Incubation of the pyruvate dehydrogenase complex with thiamin diphosphate, Mg2+, and pyruvate leads to a shortening of the lag phase, as well as to a decrease of the intrinsic tryptophan fluorescence in a time-dependent process, which evinces the same characteristics as tau. Dependence of pyruvate, as well as of the substrate analog methylacetylphosphonate, can be established by measurements of fluorescence quenching, thus ruling out an essential role of hydroxyethyl thiamin diphosphate in the process reflected by the lag phase. The results demonstrate that the lag phase is induced after the binding of both thiamin diphosphate . Mg2+ and pyruvate to the catalytic site to form a ternary enzyme complex, which undergoes subsequently a slow conformational change to an active enzyme form. This change is confined to single subunits, and no interactions between neighboring monomers could be observed. A model is proposed to describe the mechanism represented by the lag phase.     

A stopped-flow study of the reaction of cytochrome c peroxidase with hydroperoxides

Transient kinetic measurements show that cytochrome c peroxidase reacts with excess of hydroperoxides to produce compound ES in two phases. The activation energies for the fast and slow phases are calculated to be 6.3 and 20.5 kcal X mol-1, respectively. The fast phase is assigned to the reaction of native active (pulsed) cytochrome c peroxidase with peroxides, whereas the slow phase is due to the presence of an inactive (aged, resting) enzyme. As the active species is exhausted, the equilibrium between the active and inactive enzymes is shifted by a slow conformational change to replenish the active enzyme. Since the rate-limiting step of the reaction of the inactive enzyme with peroxides is the conformation change, the overall reaction rate is independent of the nature and concentration of peroxides.     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

Linkage of formate hydrogenlyase with anaerobic respiration in Proteus mirabilis

The linkage between the enzyme system catalysing formate hydrogenlyase and reductases involved in anaerobic respiration in intact cells of anaerobically grown Proteus mirabilis was studied. Reduction of nitrate and fumarate by molecular hydrogen or formate was possible under all growth conditions; reduction of tetrathionate and thiosulphate occurred only in cells harvested at late growth phase from a pH-regulated batch culture and not in cells harvested at early growth phase or in cells grown in pH-auxostat culture. Under all conditions, cells possessed the enzyme tetrathionate reductase. We conclude that linkage between tetrathionate reductase (catalysing also reduction of thiosulphate) and the formate hydrogenlyase chain is dependent on growth conditions. During reduction of high-potential oxidants such as fumarate, tetrathionate (when possible) or the artificial electron acceptor methylene blue by formate, there was no simultaneous H₂ evolution due to the formate hydrogenlyase reaction. H₂ production started only after complete reduction of methylene blue or fumarate, in the case of methylene blue after a lag phase without gas production. In preparations with a low fumarate reduction activity this was accompanied by an acceleration in CO₂ production. During reduction of thiosulphate (a low-potential oxidant) or of tetrathionate in the presence of benzyl viologen (a low-potential mediator) by formate, H₂ was evolved simultaneously. From this we conclude that formate hydrogenlyase is regulated by a factor that responds to the redox state of any electron acceptor couple present such that lyase activity is blocked when the acceptor couple is oxidised to too great an extent.     

A functionally inactive, cold-stabilized form of the Escherichia coli F1Fo ATP synthase

An unusual effect of temperature on the ATPase activity of E. coli F1Fo ATP synthase has been investigated. The rate of ATP hydrolysis by the isolated enzyme, previously kept on ice, showed a lag phase when measured at 15 degrees C, but not at 37 degrees C. A pre-incubation of the enzyme at room temperature for 5 min completely eliminated the lag phase, and resulted in a higher steady-state rate. Similar results were obtained using the isolated enzyme after incorporation into liposomes. The initial rates of ATP-dependent proton translocation, as measured by 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching, at 15 degrees C also varied according to the pre-incubation temperature. The relationship between this temperature-dependent pattern of enzyme activity, termed thermohysteresis, and pre-incubation with other agents was examined. Pre-incubation of membrane vesicles with azide and Mg2+, without exogenous ADP, resulted in almost complete inhibition of the initial rate of ATPase when assayed at 10 degrees C, but had little effect at 37 degrees C. Rates of ATP synthesis following this pre-incubation were not affected at any temperature. Azide inhibition of ATP hydrolysis by the isolated enzyme was reduced when an ATP-regenerating system was used. A gradual reactivation of azide-blocked enzyme was slowed down by the presence of phosphate in the reaction medium. The well-known Mg2+ inhibition of ATP hydrolysis was shown to be greatly enhanced at 15 degrees C relative to at 37 degrees C. The results suggest that thermohysteresis is a consequence of an inactive form of the enzyme that is stabilized by the binding of inhibitory Mg-ADP.     

On the role of molecular oxygen in lipoxygenase activation: comparison and contrast of epidermal lipoxygenase-3 with soybean lipoxygenase-1

The oxygenation of polyunsaturated fatty acids by lipoxygenases (LOX) is associated with a lag phase during which the resting ferrous enzyme is converted to the active ferric form by reaction with fatty acid hydroperoxide. Epidermal lipoxygenase-3 (eLOX3) is atypical in displaying hydroperoxide isomerase activity with fatty acid hydroperoxides through cycling of the ferrous enzyme. Yet eLOX3 is capable of dioxygenase activity, albeit with a long lag phase and need for high concentrations of hydroperoxide activator. Here, we show that higher O(2) concentration shortens the lag phase in eLOX3, although it reduces the rate of hydroperoxide consumption, effects also associated with an A451G mutation known to affect the disposition of molecular oxygen in the LOX active site. These observations are consistent with a role of O(2) in interrupting hydroperoxide isomerase cycling. Activation of eLOX3, A451G eLOX3, and soybean LOX-1 with 13-hydroperoxy-linoleic acid forms oxygenated end products, which we identified as 9R- and 9S-hydroperoxy-12S,13S-trans-epoxyoctadec-10E-enoic acids. We deduce that activation partly depends on reaction of O(2) with the intermediate of hydroperoxide cleavage, the epoxyallylic radical, giving an epoxyallylic peroxyl radical that does not further react with Fe(III)-OH; instead, it dissociates and leaves the enzyme in the activated free ferric state. eLOX3 differs from soybean LOX-1 in more tightly binding the epoxyallylic radical and having limited access to O(2) within the active site, leading to a deficiency in activation and a dominant hydroperoxide isomerase activity.