Professor Charles H. Clapp, Biochemistry
Mechanism and Inhibition of Lipoxygenases
Most of the work in the Clapp lab concerns soybean lipoxygenase, a non-heme iron protein that catalyzes the oxygenation of linoleic acid (1) to 13-hydroperoxy-9,11-octadecadienoic acid (2).
The goals of this work are to understand the catalytic mechanism of this enzyme and to discover new inhibitors of the enzyme. It is hoped that the insight derived from this work will contribute to the design inhibitors of human lipoxygenases that are thought to be involved in inflammation, asthma, and possibly atherosclerosis. Studying the soybean enzyme has the advantage that the three-dimensional structure has been determined by X-ray diffraction. In addition, the purification procedure is of reasonable length for undergraduates and introduces them to commonly used techniques (ammonium sulfate fractionation and ion-exchange chromatography). Each student in the group will purify the enzyme and then work on a project related to its basic chemistry.
One approach to probing the chemistry of this enzyme is to study the action of the enzyme on synthetic substrates that have been designed to test ideas about the catalytic mechanism. For example, we have discovered that lipoxygenase will catalyze elimination reactions on substrate analogs that have potential leaving groups on the carbon adjacent to the C-H bond that is cleaved in the normal catalytic reaction. One substance of this type, 12-iodo-cis-9-octadecenoic acid, has been found to be a mechanism-based inhibitor of lipoxygenase. We are currently studying the mechanisms of these reactions in order to better understand the mechanism by which lipoxygenase cleaves C-H bonds.
In order to determine the relative importance of the two double bonds of linoleic acid to the lipoxygenase reaction, we are studying the action of the enzyme on the two monoenoic acid analogues: 9-octadecenoic acid and 12-octadecenoic acid. We have found that both of these substances are substrates for the enzyme. 9-Octadecenoic acid is slowly oxygenated to 11-oxo-9-octadecenoic acid, and 12-octadecenoic acid gives two major products, which will be identified by future students. Future studies will involve studying the kinetics of these reactions and attempting to trap or detect possible intermediates.
Typical student projects involve (1) synthesis of novel substrates of the enzyme, (2) isolation and characterization of the products from novel lipoxygenase substrates, (3) design, synthesis and testing of new inhibitors, (4) kinetic studies using both spectroscopic and HPLC methods, and (5) studies on the interaction of inhibitors with the enzyme using electron spin resonance and fluorescence spectroscopy.
Department of Chemistry,