Stopped-Flow Kinetic Investigations of the Activation of Soybean Lipoxygenase-1 and the Influence of Inhibitors on the Allosteric Site
- 22 March 2003
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 42 (14) , 4172-4178
- https://doi.org/10.1021/bi020698o
Abstract
Herein, we report on the role of the allosteric site in the activation mechanism of soybean lipoxygenase-1 utilizing stopped-flow inhibition kinetic studies. The KD for the activation was determined to be 25.9 ± 2.3 μM and the rate constant for the oxidation of the iron cofactor, k2, to be 182 ± 4 s-1. Two inhibitors were employed in this study, (Z)-9-octadecenyl sulfate (OS) and (Z)-9-palmitoleyl sulfate (PS), of which OS is an allosteric inhibitor of the turnover process, while PS is a linear mixed inhibitor with a Ki of 13.7 ± 1.3 μM for the catalytic site and a Ki‘of 140 ± 9 μM for the allosteric site. It was found that OS does not inhibit the activation of soybean lipoxygenase-1, while PS acts as a competitive inhibitor versus the product, 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, with a Ki of 17.5 ± 3.8 μM. These results suggest that OS binds to an allosteric site that is separate from the catalytic iron site. We further observed that the allosteric site binding selectivity is sensitive to inhibitor length as seen by its preference for OS over that of PS, which is two carbons longer than PS.Keywords
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