Determination of the Lid Residues Critical for Catalysis by Cholestrol Oxidase

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Sampson 9405394 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 10A, 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 11A 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 A 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.) 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 questions that will be addressed are: (1) what is the mechanism of the oxidative process and is it the rate-limiting step in the enzymatic reaction? (2) if we substitute side-chains of the surface loops with alanine by site-directed mutagenesis, what happens to the ability of the enzyme to sequester substrates and intermediates and to catalyze their transformation? The consequences of the alanine-substitution mutations on the following properties will be investigated: (i) the catalytic rate and on the rate-limiting step, using kinetic isotope effects. (ii) the oxidative process, by studying the effects of (a) deazaFAD reconstitution (b) cyclopropyl radical traps (c) mutagenizing the putative general base. (iii) the affinity for and the turnover of hydroxysteroids with various alkyl C-17 tails. 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 effects 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. %%% Arteriosclerosis is a widespread disease amongst the American population. This coronary disease is partly a result of high levels of cholesterol in the blood. One way to reduce the incidence of this disease is to monitor the blood cholesterol levels of individuals and to prescribe measures to reduce cholesterol levels for those with unhealthy levels of cholesterol. Our research will lead to the development of more facile and inexpensive tests of blood cholesterol levels, that will lead to easier prescription of preventative measures. The goal of the proposed research is to study how the structure of the protein catalyst, cholesterol oxidase, relates to function. From the structural information that is available, it is not clear how the substrate, cholesterol, binds to the catalyst or how it is released. It has been proposed that two fragments of the catalyst are essential for binding and release of cholesterol. We will modify the structure of t hese fragments using molecular biological techniques. The modifications affect binding and release of cholesterol, use assays of serum cholesterol levels. In addition, this study will lead to basic insight into the mechanisms of steroid (for example, cholesterol) transformation and transport in the body. It may be possible in the long term to develop new biomaterials using information obtained from this type of study. The interaction of the protein with the lipid interface is not well understood. Two dimensional lipid interfaces may be constructed with novel proteins attached to them which will allow the development of biosensors for cholesterol or other steroids. These biomaterials may be more stable and less susceptible to biological degradation than some materials currently available. ***