In this award from the Chemistry of Life Processes Program of the Division of Chemistry, Dr. Charles Clapp, from Bucknell University, will use chemical synthesis and site-directed mutagenesis to investigate the binding of substrates to the enzyme soybean lipoxygenase-1, with a focus on the role of phenylalanine-557 (phe-557), which has been proposed to be at the binding site for the methyl terminus of substrates. This hypothesis will be tested by synthesizing fatty acids that are slightly longer than linoleic acid and determining whether these new substrates show enhanced activity with site-directed mutants in which phe-557 is replaced with smaller amino acids. It has also been proposed that replacing phe-557 with smaller amino acids alters the regiochemistry of the reaction by allowing substrates to bind in a reverse manner. This hypothesis will be tested using synthetic substrates, stereochemical studies, and additional mutagenesis.
Lipoxygenases catalyze a useful reaction, the incorporation of oxygen into polyunsaturated fatty acids, that chemists cannot carry out in a controlled way without enzymes. Understanding the catalytic mechanism of these enzymes provides an opportunity to learn useful chemistry from nature. Soybean lipoxygenase-1 is the most extensively studied lipoxygenase, but our mechanistic insight is limited by uncertainty about how substrates bind. The proposed work is designed to address this deficiency. Better understanding of how lipoxygenases bind substrates could lead to the rational design of mutant lipoxygenases that would catalyze transformations that cannot be achieved with the natural enzymes. Undergraduate students will play a major role in this work. The proposed experiments use basic techniques that students learn about in their courses, and students working on this project will gain experience applying these techniques to test hypotheses in research.
