Date of Thesis

2013

Description

Soybean lipoxygenase-1 is a model for lipoxygenase activity. While the mechanism of oxygenation is understood, the substrate binding mechanism has not yet been elucidated. Two putative binding mechanisms are the ¿head-first¿ and ¿tail-first¿ models, in which the carboxy-terminus or the methyl terminus of the fatty acid substrate is inserted into the active site while the remainder of the molecule protrudes from the surface, respectively. Previous work has demonstrated that derivatization of fatty acid substrates with D-tryptophan increases active site affinity. It has also been shown that while polyunsaturated fatty acids are the natural substrates of lipoxygenases, monounsaturated fatty acids can be oxygenated at a much slower rate. Starting with a monounsaturated fatty acid, oleic acid, as a platform, the molecule N-oleoyl-D-tryptophan (ODT) was synthesized with the anticipation of it being a potent competitive substrate-analogue inhibitor that could be used to discern the substrate binding mechanism. Inhibition kinetics demonstrated that this molecule functions as a partially competitive inhibitor, through an unknown mechanism. The implication behind partially competitive inhibition is that substrate and inhibitor molecules can bind simultaneously to the enzyme, which alludes to the presence of an allosteric binding domain. To investigate the possibility of an inhibitor binding site on the non-catalytic subunit, limited proteolysis was used to cleave the subunits apart which should have eliminated inhibition. Interestingly, it was observed that at high substrate concentrations the inhibitor was completely ineffective, but at low substrate concentrations the inhibitor maintained its standard efficacy. A satisfactory explanation for these results has not yet been determined.

Keywords

Enzyme, Protein, Lipoxygenase, Kinetics, Substrate, Inhibitor

Access Type

Honors Thesis (Bucknell Access Only)

Degree Type

Bachelor of Science

Major

Chemistry

First Advisor

Charles Clapp

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