Lipoxygenases are implicated as key biological players in asthma, heart disease and cancer. They are pharmaceutical targets and have lead to the discovery of various therapeutic treatments. The current proposal encompasses four aims of study in order to establish a better understanding of the enzymatic mechanism for lipoxygenase and to discover and characterize selective inhibitors.1) The general mechanistic scheme for soybean lipoxygenase- 1 (SLO) is well understood but the molecular details of the individual steps remain unclear. In the current proposal, we plan to determine the molecular mechanism of SLO using time-resolved crystallography, stopped-flow spectroscopy and steady-state kinetics to probe the active site base (Fe(III-OH-) and the molecular mechanism for hydrogen bond rearrangement.2) Although our understanding of SLO is maturing rapidly, our knowledge of the human platelet 12-lipoxygenase (12-HLO) and human reticulocyte 1 5-lipoxygenase (15-HLO) remains limited, even though they are the targets for therapeutic treatment. We therefore plan to extend our mechanistic studies of SLO to include the human enzymes which will allow us to answer the fundamental question; how do the structural differences between the three lipoxygenases affect their function?3) Over the past several years, we have proposed the presence of an allosteric site in both SLO and 15-HLO, which could be critical to the regulation of lipoxygenase activity, however its location remains unknown. We have therefore proposed to co-crystallize lipoxygenase with our allosteric inhibitors and mutate the putative binding site to establish the location and binding constraints of the site. We shall also employ inhibitor displacement experiments and stopped-flow fluorescence kinetics to establish the enzymatic role of the allosteric site and its effect on catalysis.4) Finally, we shall continue to discover novel lipoxygenase inhibitors by screening a marine natural products (MNP) library (12 inhibitors from about 6OO extracts in the last 24 months). We will eventually establish an inhibitor library that will be investigated with biochemical and spectroscopic methods to determine how the compounds bind and inhibit lipoxygenase. This will allow us to draw structural trends and design the next generation of inhibitors, which will selectively inhibit 12-HLO or 15-HLO and target either the catalytic or allosteric sites.
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