The interactions of cholesterol oxidase with its substrate and with the lipid bilayer will be explored in order to understand its substrate specificity. Cholesterol oxidase is a flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The chemistry catalyzed is well understood, yet structural knowledge of the catalytically active enzyme is at very low resolution. A model of the catalytically-active complex is described. The specific issues that will be addressed in the next grant period are: l. What happens to the hydrogen bond networks during catalysis? 2. What is the orientation of the enzyme on the membrane surface? 3. What is the conformation of the protein when cholesterol is bound? Does the active site lid undergo a rigid body hinge-like movement, or does the lid structure rearrange to pack with the steroid tail? 4. Using existing knowledge of the structure, can the substrate specificity of cholesterol oxidase be altered to develop mutants that are more specific for plant sterols than cholesterol? 5. Does the activity of cholesterol oxidase directly depend on lipid phase? Thus, the proposed experiments are aimed at elucidating the structure of the active complex of enzyme and membrane. A combination of high resolution X-ray crystallography, spectroscopy, and kinetics will be used. Cholesterol oxidase is used commercially for the determination of serum cholesterol concentrations, and it is being developed as an insecticide by the agribiochemical industry. Moreover, cholesterol oxidase is used to study the localization of cholesterol in membranes. These studies will provide a precise molecular model of the active enzyme complexed with steroid substrate. This model is important for the development of cholesterol oxidase both as an effective commercial product and as a useful tool in the study of cellular membranes, i.e., understanding the structure of lipid 'rafts' that have been implicated in cellular signaling.