INVESTIGATION OF MEMBRANE DISRUPTION IN THE REACTION CATALYZED BY CHOLESTEROL OXIDASE

Dye leakage experiments were undertaken to investigate the membrane di sruption properties of cholesterol oxidase. Inspection of the X-ray cr ystal structures of cholesterol oxidase suggested that an active-site ''lid'' opens in order to bind substrate [Li, J., Vrielink, A., Brick, P., & Blow, D. M. (1993) Biochemistry 32, 11507-11515]. We tested whe ther the interaction of the putative active-site lid with the membrane was sufficiently disruptive of the membrane structure to cause leakag e or lysis of the cell membrane. Vesicles (100 nm) composed of egg pho sphatidylcholine, 2-palmitoyl-3-oleoyl- 1-sn-phosphatidylethanolamine, and 2-palmitoyl-3-oleoyl- 1 -sn-phosphatidylcholine were used in this study to mimic biomembranes. To separate the effects of membrane bind ing from conversion of cholesterol to cholest-4-en-3-one, the active-s ite mutant E361Q was utilized. In the reaction catalyzed by E361Q, iso merization of the cholest-5-en-3-one intermediate is suppressed and ch olest-5-en-3-one is the major product isolated. Furthermore, E361Q pro duces cholest-5-en-3-one 20-fold more slowly than wild type produces c holest-4-en-3-one from cholesterol. Wild-type and E361Q cholesterol ox idases bind to vesicles with an apparent K-D of approximately 25 mu M, as measured by quenching of intrinsic tryptophan fluorescence, irresp ective of headgroup size and cholesterol content. Membrane disruption was measured by leakage of the encapsulated marker carboxyfluorescein. Leakage was observed with cholesterol containing vesicles and wild-ty pe enzyme only; the rate of leakage was dependent on the rate of chole st-4-en-3-one production. E361Q did not induce membrane disruption, re gardless of vesicle type tested. Thus, binding of cholesterol oxidase to the membrane and partitioning of cholesterol into the active site d oes not sufficiently perturb the bilayer to cause leakage of vesicle c ontents. Formation of the product cholest-4-en-3-one, however, does in crease membrane permeability. Expansion of the lipid bilayer upon conv ersion of cholesterol to cholest-4-en-3-one is the likely cause of thi s increased permeability.