Doctor of Philosophy (PhD)
Leukotrienes are potent immune-modulatory compounds involved in the inflammatory response. 5-Lipoxygenase (5-LOX) is the enzyme that catalyzes the production of Leukotriene A4 (LTA4), which is further metabolized to pro- and anti-inflammatory compounds derived from the substrate Arachidonic Acid (AA), a polyunsaturated fatty acid. The crystal structure of a stabilized form of 5-LOX has been recently reported (Gilbert, Bartlett et al. 2011). However, the structure does not reveal the mode of substrate entry or recognition. The first step in the enzymatic reaction involves the removal of a hydrogen from the central carbon of a pentadiene present in the substrate. It is not known how 5-LOX distinguishes among three such chemically equivalent pentadienes in AA to produce only 5-S-HPETE, which is subsequently transformed to LTA4. The crystal structure suggests a somewhat crescent shaped active site lined with invariant Leucines and Isoleucines, with no clear access port visible for substrate entry. A series of site-directed mutants were produced and the activities of these mutants evaluated. Product analyses of these mutants with AA and substrate analogs suggest entry of the substrate by the opening of the so-called Phenylalanine (F)-Tyrosine(Y) ‘cork’. The data is also consistent with the orientation of AA as carboxyl innermost, as predicted from the stereochemistry of the product and mechanistic models of lipoxygenase function.
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Mitra, Sunayana, "Insights into 5-Lipoxygenase Active Site and Catalysis" (2015). LSU Doctoral Dissertations. 3000.