Use of the Parallax-Quench Method to Determine the Position of the Active-Site Loop of Cholesterol Oxidase in Lipid Bilayers

Abstract

To elucidate the cholesterol oxidase-membrane bilayer interaction, a cysteine was introduced into the active site lid at position-81 using the Brevibacterium enzyme. To eliminate the possibility of labeling native cysteine, the single cysteine in the wild-type enzyme was mutated to a serine without any change in activity. The loop-cysteine mutant was then labeled with acrylodan, an environment-sensitive fluorescence probe. The fluorescence increased and blue-shifted upon binding to lipid vesicles, consistent with a change into a more hydrophobic, i.e., lipid, environment. This acrylodan-labeled cholesterol oxidase was used to explore the pH, ionic strength, and headgroup dependence of binding. Between pH 6 and 10, there was no significant change in binding affinity. Incorporation of anionic lipids (phosphatidylserine) into the vesicles did not increase the binding affinity nor did altering the ionic strength. These experiments suggested that the interactions are primarily driven by hydrophobic effects not ionic effects. Using vesicles doped with either 5-doxyl phosphatidylcholine, 10-doxyl phosphatidylcholine, or phosphatidyl-tempocholine, quenching of acrylodan fluorescence was observed upon binding. Using the parallax method of London [Chattopadhyay, A., and London, E. (1987) Biochemistry 26, 39-45], the acrylodan ring is calculated to be 8.1 +/- 2.5 A from the center of the lipid bilayer. Modeling the acrylodan-cysteine residue as an extended chain suggests that the backbone of the loop does not penetrate into the lipid bilayer but interacts with the headgroups, i.e., the choline. These results demonstrate that cholesterol oxidase interacts directly with the lipid bilayer and sits on the surface of the membrane.

How cholesterol homeostasis is regulated by plasma membrane cholesterol in excess of phospholipids

“…Crystallographic studies suggest that cholesterol oxidase accesses substrate sterol molecules as they transiently project from the bilayer surface (27). The high activity of plasma membrane cholesterol above the threshold may reflect the frequency of such projections (18,26).…”

Stability and Stoichiometry of Bilayer Phospholipid–Cholesterol Complexes: Relationship to Cellular Sterol Distribution and Homeostasis

“…Polar head group species did not seem to exert a strong influence; in contrast, the RA values above threshold were orders of magnitude greater for unsaturated than saturated chains. The large differences in enzyme kinetics among the LUVs probably do not represent variations in K d , the disassociation constant for the binding of cholesterol oxidase, given that (a) the enzyme binds superficially to the surface of the vesicles; (b) the K d is rather insensitive to vesicle composition; and (c) the various phosphorylcholine phospholipids should bind the enzyme similarly yet had widely divergent RA values (38, 42, 43). …”

“…(62). ] Furthermore, the Michaelis, dissociation and catalytic constants (K m , K d and k cat ) of the enzyme should not vary with the cholesterol content of the bilayer (38, 39, 42, 43). We also considered whether the observed thresholds might reflect the limited solubility of the cholesterol, since sterols in excess of a sharp threshold form crystals in phospholipid bilayers (61).…”

Cholesterol homeostasis and the escape tendency (activity) of plasma membrane cholesterol

“…Structural studies have shown that this enzyme associates with the surface of the bilayer rather than penetrating into its nonpolar core. Projecting sterol molecules can just reach the catalytic cavity of the periphally-disposed enzyme [74,75]. The catalytic rate of cholesterol oxidase can therefore report on the constraints imposed by various phospholipids on the escape tendency (bobbing activity) of the sterols [57].…”