Evaluation of the Role of His⁴⁴⁷in the Reaction Catalyzed by Cholesterol Oxidase^†

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one via cholest-5-en-3-one. It has been proposed that His(447) acts as the general base catalyst for oxidation, and that the resulting imidazolium ion formed acts as an electrophile for isomerization, In this work, we undertook an assessment of the proposed dual roles of His(447) in the oxidation and isomerization reactions. To test its role, we constructed five mutants, H447Q, H447N, H447E, H447D, and H447K, that introduce hydrogen bond donors and accepters and carboxylate bases at this position, and a sixth mutant, E361Q, to test: the interplay between His(447) and Glu(361). These mutants were characterized using steady-state kinetics and deuterium substrate and solvent isotope effects. For those mutants that catalyze either oxidation of cholesterol or isomerization of cholest-5-en-3-one, the K-m's vary no more than 3-fold relative to wild type. H447K is inactive in both oxidation (>100 000-fold reduced) and isomerization assays (>10 000-fold reduced). H447E and H447D do not catalyze oxidation (>100 000-fold reduced), but do catalyze isomerization, 10(4) times slower than wild type. The k(cat) for H447Q is 120-fold lower than wild type for oxidation, and the same as wild type for isomerization. The k(cat) for H447N is 4400-fold lower than wild type for oxidation, and is 30-fold lower than wild type for isomerization. E361Q does not catalyze isomerization (>10 000-fold reduced), and the k(cat) for oxidation is 30-fold lower than wild type. The substrate deuterium kinetic isotope effects for the wild-type and mutant-catalyzed oxidation reactions suggest that mutation of His(447) to an amide results in a change of the rate-determining step from hydride transfer to hydroxyl deprotonation. The deuterium solvent and substrate kinetic isotope effects for isomerization indicate that an amide at position 447 is an effective electrophile to catalyze formation of a dienolic intermediate. Moreover, consideration of kinetic and structural results together suggests that a hydrogen bonding network involving His(447), Glu(361) and Asn(485), Wat(541), and substrate serves to position the substrate and coordinate general base and electrophilic catalysis, That is, in addition to its previously demonstrated role as base for deprotonation of carbon-4 during isomerization, Glu(361) has a structural role and may act as a general base during oxidation, The His(447), Asn(485), Glu(361), and Wat(541) residues are conserved in other GMC oxidoreductases. Observation of this catalytic tetrad in flavoproteins of unknown function may be diagnostic for an ability to oxidize unactivated alcohols.