UC Santa Cruz

The research contained in this dissertation examines the kinetic, mechanistic, and structural basis of human lipoxygenase (LOX) catalysis. LOX are found throughout the plant and animals kingdoms, and in humans LOX are involved in inflammatory signaling. Through this involvement in the inflammatory process and the ability to oxidize membrane bound polyunsaturated fatty acids (PUFA), LOX have been implicated in a variety of cancers and disease states. The impact of substrate saturation on platelet 12-LOX kinetics and mechanism was investigated in Chapter 2. The platelet signaling pathways and implications on platelet aggregation of these substrates were determined. Additionally, the novel allosteric effect of 15S-HpEPE on 12-LOX mechanism was probed. Chapter 3 scrutinized a previously discovered lipoxygenase activator. This dissertation works shows that the activator is LOX isoform specific, with only h15-LOX-1 being kinetically activated in the presence of the compound. Furthermore, the activator was found to activate the catalysis of shorter PUFA substrates, AA and LA, but not that of the longer SPM precursor, DHA. The implications of this finding has consequences on the potential therapeutic development of the activating compound. The effect on enzyme kinetics and mechanism was assayed, and a V-type activation was observed. Chapter 4 builds off of a previous discovery in our lab that demonstrated the altered positional specificity of h15-LOX-1 when reacting with the metabolites of 5-LOX. In this chapter, site-directed mutagenesis was performed on key active site residues, with the kinetic and mechanistic implications of those residues determined experimentally. Additionally, the consequence of the altered positional specificity with regards to the biosynthesis of the potent SPM, Resolvin E4, was examined.