Date of Thesis
Lipoxygenases are a class of enzymes found in plants, animals, fungi, and bacteria that catalyze essential biological reactions. Soybean lipoxygenase-1 (SBLO-1) utilizes a non-heme iron to catalyze the oxygenation of a cis double bond to a conjugated diene hydroperoxide. The structure of SBLO-1 is known, but the exact substrate binding mechanism is not. There are several theories to explain substrate binding, two of which are considered here. The first hypothesizes that the methyl terminus of fatty acid substrate enters the binding pocket of the enzyme, leaving the charged head group exposed but that larger substrate derivatives bind differently. The kinetics of serine-491 mutants were studied to probe this hypothesis. S491 was hypothesized to form a hydrogen bond with the carboxy-terminus of substrate. Mutations to alanine (S491A), valine (S491V), and methionine (S491M) removed the potential for hydrogen bonds, as well as introducing size constraints in the valine and methionine mutants. Kinetic rate losses were observed, but the differences in rate were smaller than expected for a hydrogen bond between enzyme and substrate. To test the hypothesis that larger substrates bind differently, the kinetics of S491M with linoleoyl-L-tryptophan (lin-L-trp), a linoleic acid derivative with the carboxylate group attached to a tryptophan residue, were studied. Decreases in Km and Vmax were observed, but more recent findings suggest that the lin-L-trp concentration was above the critical micelle concentration (CMC). Thus the kinetics observed may not be indicative of free substrate but rather exposed tails of micelles. The second hypothesis explored in this work concerns the transient separation of Â¿-helices 2 and 11 to allow for substrate entry to the active site. Previous work in this lab generated a mutant containing cysteine residues on both helices, T259C,S545C, for formation of a disulfide linkage. A mobility shift in SDS-PAGE analysis is observed under aerobic conditions at pH 9.0 and at pH 9.2, suggesting formation of a disulfide bond. Mass spectrometry (MS) analysis was performed on trypsin digests of the T259C,S545C enzyme to determine the presence of a disulfide. Under the digestion conditions used there is no observed evidence for the presence of the disulfide or reduced helix 11. Future work will focus on different digestion conditions to obtain optimal MS results.
Honors Thesis (Bucknell Access Only)
Bachelor of Science
Charles H. Clapp
Tolbert, Audrey Elizabeth, "Studies with Site-Directed Mutants to Probe Substrate Binding by soybean lipoxygenase-1" (2015). Honors Theses. 323.