On the mechanism of action of soybean lipoxygenase-1. A stopped-flow kinetic study of the formation and conversion of yellow and purple enzyme species

Abstract

1. The conversion of the native iron-containing lipoxygenase-1 into yellow and purple ferric enzyme species by 13-L-hydroperoxy-9-cis, 11-trans-octadecadienoic acid (R-13-OOH) was studied by measuring the absorbance changes at 330 nm and 580 nm, respectively in the stopped-flow apparatus. The pseudo-first-order rate constant for this reaction was found to be 50 s1 at 4.4°C, either in the presence or absence of O2. This rate constant is lower than the one for R-13-OOH formation from linoleic acid and O2 catalysed by lipoxygenase-1: kcat = 232 s1 at 4.4°C.

2. The coloured ferric enzymes are formed only in the presence of R-13-OOH; therefore the formation of the coloured ferric enzymes from oxygen, linoleic acid and the native enzyme indicates that the latter is active even before it is converted into the ferric forms.

3. Under anaerobic conditions the yellow and purple enzyme species are rapidly converted into colourless ferrous enzyme species by linoleic acid at a rate which is faster than the formation of the coloured enzymes from R-13-OOH and the native ferrous enzyme.

4. Both the aerobic formation of the coloured ferric enzymes and the anaerobic bleaching of these enzymes occur more slowly when [11-2H2]linoleic acid instead of unlabeled linoleic acid is used. The kinetic deuterium isotope effect as estimated from the rates of aerobic formation of R-13-OOH is approximately 9, whereas in the anaerobic conversion of R-13-OOH and deuterated linoleic acid a value of 1.1 is found. This demonstrates that H abstraction from linoleic acid determines the rate of the aerobic formation but not of the anaerobic conversion of R-13-OOH.

5. The rate constant for the anaerobic conversion of [11-2H2]linoleic acid by the yellow ferric enzyme species, k(2H) = 30 s1 at 25°C, is close to the rate constant for the overall oxygenation reaction of the deuterated substrate, kcat(2H) = 32 s1. For unlabelled linoleic acid the rate constants of the anaerobic H-abstraction step and the overall oxygenation reaction were found to be 111 s-1 and 290 s1, respectively at 25°C. The former rate constant is probably affected by substrate inhibition.

6. Since the native ferrous enzyme is capable of catalysing H abstraction from linoleic acid under aerobic but not under anaerobic conditions, while the yellow ferric enzyme is active either in the absence or presence of O2, it is proposed that O2 reversibly converts the ferrous enzyme species into a ferric state.