An intriguing problem of interfacial catalysis is exemplified by the enzyme cholesterol oxidase. This water soluble enzyme extracts cholesterol out of the lipid membrane bilayer, with a net movement of approximately 10 Angstroms, into a deep active site pocket. The substrate is oxidized and isomerized, and the resulting ketone is returned to the lipid bilayer. There is no obvious pathway, however, for the substrate to reach the active site. Examination of the X-ray crystal structure reveals an active site that is 11 Angstroms long (suitable for binding cholesterol), adjacent to the FAD cofactor, and closed off from solvent by two surface loops (5 and 20 residues long). (The dehydroisoandrosterone bound structure reveals a 1-2 Angstrom movement in one of the loops to accommodate the steroid in the binding site; cholesterol presumably causes a larger rearrangement with its C-17 tail.[2, 3]) It is postulated that these two loops must open to expose the hydrophobic active site to the substrate once the oxidase has diffused to the lipid membrane surface. The goal of this proposed research is to determine what structural elements are necessary for movement, binding, and catalysis to occur. A combination of site-directed mutagenesis studies and mechanistic experiments using substrate analogs will address the role of surface loops in binding substrate and product. Studies to determine the relative stabilities of enzyme-bound species promise to identify the components of the enzyme structure necessary for intermediate stabilization. Furthermore, construction of liposomes containing substrate and substrate analogs allows binding phenomena at the membrane interface to be investigated. All of these experiments will lead to a model for binding and catalysis at the two-dimensional lipid interface. This model will be relevant to understanding the mode of action of other steroid binding proteins and enzymes, for example, the enzymes required for the biosynthesis of steroids and proteins involved in sterol transport. Furthermore-, cholesterol oxidase is used extensively in clinical applications for the determination of serum cholesterol levels. Understanding how the structure effects catalysis will result in the design of a cholesterol oxidase more suitable for immobilization and clinical assay purposes. How mechanical conformational changes in proteins effect binding and catalysis will be better understood as a result of the proposed research. The capability to alter and modify enzyme function for a specific purpose is still in the infant phases of development, and a set of general rules for creating structure and function is only beginning to emerge from the wide range of observations that have been made. It is with the type of detailed study outlined in this proposal that these rules will become more apparent.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
First Independent Research Support & Transition (FIRST) Awards (R29)
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Bio-Organic and Natural Products Chemistry Study Section (BNP)
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State University New York Stony Brook
Schools of Arts and Sciences
Stony Brook
United States
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