Allosteric, mechanistic, and inhibitory investigations of human platelet 12-lipoxygenase, human reticulocyte 15-lipoxygenase-1 and human epithelial 15-lipoxygenase-2.

摘要

The research presented in this dissertation is centered on allosteric investigations of lipoxygenase (LO). Human lipoxygenases (hLO) are an important class of metallo-enzymes involved in regulating inflammation response by initiated oxylipid signaling cascades via their hydroperoxide metabolites of arachidonic acid (AA) and linoleic acid (LA). There is growing evidence demonstrating hLO involvement in many inflammatory diseases and a variety of cancer, making the LO family a suitable pharmacological target. Recent data has suggested the substrate specificity of these enzymes for either arachidonic acid or linoleic acid, may play a role in cancer progress since the LO metabolites of these fatty acids have different cellular responses. Therefore, determining the substrate specificity of these enzymes is critical in understanding there role in cancer progression.;This dissertation investigates the allostery of lipoxygenase using three distinct methods: substrate specificity studies, mechanistic studies and inhibition studies. Using a novel competitive substrate capture experiment which measures the simultaneous product formation of a two substrate enzyme assay, we have discovered an intricate product-feedback mechanism that differentially affects the substrate specificity of the 15-hLO isozymes. Probing the allostery using isopote effect experiments, we have demonstrated that allosteric binding can affect the rate-limiting contributions from the individual steps in the catalytic mechanism of 15-hLO, suggestive of a regulatory mechanism for substrate specificity. Finally, inhibition studies have demonstrated allosteric inhibitors not only suppress catalytic activity, but also affect substrate selectivity. These findings are significant for drug discovery, introducing a new approach for manipulating hLO activity in inflammatory diseases and cancer.