Catalysis at the Membrane Interface: Cholesterol Oxidase as a Case Study

Interfacial enzymes present additional challenges in their study compared to enzymes with soluble substrates. Cholesterol oxidase is an interfacial enzyme that transiently associates with lipid membranes to convert cholesterol to cholest-4-en-3-one. As a case study to exemplify the issues that should be considered, we describe our structural and mechanistic understanding of cholesterol oxidase kinetic activity based on X-ray crystal structures and kinetic analysis. Introduction Interfacial enzymes are water-soluble enzymes that catalyze reactions with membrane-soluble substrates. Kinetic characterization of these enzymes is made more complex by the necessity to consider the role of the interface and interactions with the interface in assessing the catalytic activity. Moreover, the interface can change during catalysis, further complicating the kinetic analysis. The interface used in assaying the enzyme influences the apparent substrate specificity measured. Thus, the assignment of a physiological role for an enzyme is dependent on the interface employed in enzymatic assays. Cholesterol oxidase is one such water-soluble enzyme that is catalytically active at the membrane interface from which cholesterol, the substrate, is accessed. As a case study, we present work from our laboratory that characterizes what happens at the membrane interface. We delineate the kinetic issues in reporting the catalytic activity of such an enzyme. 13 http://www.beilstein-institut.de/escec2007/proceedings/Sampson/Sampson.pdf ESCEC, September 23 – 26, 2007, Rüdesheim/Rhein, Germany