Oxygen Access to the Active Site of Cholesterol Oxidase through a Narrow Channel Is Gated by an Arg-Glu Pair

• Published in: The Journal of biological chemistry Vol. 276; no. 32; pp. 30435 - 30441
• Main Authors: Coulombe, R, Yue, K Q, Ghisla, S, Vrielink, A
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 10-08-2001
• Subjects: Arginine - chemistry; Binding Sites; Brevibacterium - enzymology; Cholesterol - chemistry; Cholesterol Oxidase - chemistry; Cholesterol Oxidase - metabolism; Electrons; Flavins - chemistry; Glutamic Acid - chemistry; Kinetics; Models, Chemical; Models, Molecular; Oxygen - metabolism; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M104103200

Cholesterol oxidase is a monomeric flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one.
Two forms of the enzyme are known, one containing the cofactor non-covalently bound to the protein and one in which the cofactor
is covalently linked to a histidine residue. The x-ray structure of the enzyme from Brevibacterium sterolicum containing covalently bound FAD has been determined and refined to 1.7-Å resolution. The active site consists of a cavity
sealed off from the exterior of the protein. A model for the steroid substrate, cholesterol, can be positioned in the pocket
revealing the structural factors that result in different substrate binding affinities between the two known forms of the
enzyme. The structure suggests that Glu 475 , located at the active site cavity, may act as the base for both the oxidation and the isomerization steps of the catalytic
reaction. A water-filled channel extending toward the flavin moiety, inside the substrate-binding cavity, may act as the entry
point for molecular oxygen for the oxidative half-reaction. An arginine and a glutamate residue at the active site, found
in two conformations are proposed to control oxygen access to the cavity from the channel. These concerted side chain movements
provide an explanation for the biphasic mode of reaction with dioxygen and the ping-pong kinetic mechanism exhibited by the
enzyme.