The objectives of this proposal is to understand the mechanism whereby hydrophilic soluble proteins interact with membranes, specifically the membrane lipid component. Recent work indicates that many such interactions exist and play a multitude of cell roles such as sensory transduction. However, little is known about the mechanisms involved. Two enzyme systems will be studied. Both are enzymes of the bacterium, Escherichia coli. E. coli pyruvate oxidase is an enzyme greatly activated (500-fold in Kcat) upon binding to lipids, but is a soluble protein closely related to certain enzymes of amino acids biosynthesis. Prior work has suggested a model for this interaction whereby the C- termini of the enzyme penetrates into the lipid bilayer and this model shall be tested by biochemical and genetic means. Development of a new method to assay penetration of proteins into a lipid bilayer is proposed. The second enzyme is E. coli cyclopropane fatty acid (CFA) synthase, a novel enzyme that modifies the double bonds of phospholipids resident in a lipid bilayer. CFA synthase methylenates the double bond to form a cyclopropane ring, the methylene donor being S-adenosyl methionine. The purified enzyme is now available and its interaction with phospholipids can now be studied in detail.
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