Evaluation of the role of His(447) in the reaction catalyzed by cholesterol oxidase

Cholesterol oxidase catalyzes the oxidation and isomerization of cholestero l to cholest-4-en-3-one via cholest-5-en-3-one. It has been proposed that H is(447) acts as the general base catalyst for oxidation, and that the resul ting imidazolium ion formed acts as an electrophile for isomerization, In t his work, we undertook an assessment of the proposed dual roles of His(447) in the oxidation and isomerization reactions. To test its role, we constru cted five mutants, H447Q, H447N, H447E, H447D, and H447K, that introduce hy drogen bond donors and accepters and carboxylate bases at this position, an d a sixth mutant, E361Q, to test: the interplay between His(447) and Glu(36 1). These mutants were characterized using steady-state kinetics and deuter ium 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-f old reduced). H447E and H447D do not catalyze oxidation (>100 000-fold redu ced), 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 s ame 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 isom erization. E361Q does not catalyze isomerization (>10 000-fold reduced), an d the k(cat) for oxidation is 30-fold lower than wild type. The substrate d euterium kinetic isotope effects for the wild-type and mutant-catalyzed oxi dation reactions suggest that mutation of His(447) to an amide results in a change of the rate-determining step from hydride transfer to hydroxyl depr otonation. The deuterium solvent and substrate kinetic isotope effects for isomerization indicate that an amide at position 447 is an effective electr ophile to catalyze formation of a dienolic intermediate. Moreover, consider ation of kinetic and structural results together suggests that a hydrogen b onding network involving His(447), Glu(361) and Asn(485), Wat(541), and sub strate serves to position the substrate and coordinate general base and ele ctrophilic catalysis, That is, in addition to its previously demonstrated r ole as base for deprotonation of carbon-4 during isomerization, Glu(361) ha s 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 al cohols.