A stopped-flow kinetic study will be conducted on soybean lipoxygenase-1, one a class of enzymes occurring in plants and mammals. These enzymes catalyze dioxygenation of polyunsaturated fatty acids possessing cis,cis-1,4-pentadiene unit(s), such as the nutritionally essential linoleic and arachidonic acids, to give cis, trans conjugated diene hydroperoxides. The native soybean enzyme, which contains a tightly bound non-heme ferrous iron, exhibits a distinctive kinetic lag phase which is abolished by the hydroperoxide product. The product has independently been shown to convert the ferrous form of the enzyme to the ferric, which unexpectedly also exhibits a lag phase. A hypothesis is advanced in this proposal to explain this apparent identity in kinetic behavior of the two enzyme forms. The proposed Scheme stipulates that the enzyme is active only in the ferric, which unexpectedly also exhibits a lag phase. A hypothesis is advanced in this proposal to explain this apparent identity in kenetic behavior of the two enzyme forms. The proposed Scheme stipulates that the enzyme is active only in the ferric oxidation state, but that it is occasionally converted to the ferrous oxidation state by leakage of the substrate derived radical into solution during turnover, necessitating reoxidation by the product to the ferric form. The major prediction of the Scheme is that an initial burst of product formation, preceding the lag phase, will be revealed only when the ferric form of the enzyme is used at high concentrations, and when the reaction is examined on a stopped-flow time scale. I have already detected this burst phase in a preliminary study; with refinement, our methodology may prove useful as quantitative diagnostic test for radical leakage by other lipoxygenases. The amount of product formed in the burst phase should equal the ratio: substrate molecules processed/radicals located into solution. To test whether this equality is true, a number of chemical traps will be employed in quantifying substrate radicals. Additional studies will involve experiments to determine whether a hydrogen atom or a proton is lost in the rate determining step in catalysis; and synthesis of peroxides, other than the natural product, as alternate activators that may be useful in brobing the mechanism of the enzyme. The long term goal of this project is to design mechanism-based inactivators of lipoxygenase tha may prove to be of biomedical importance. Two potential inactivators will be synthesized and tested.