Functional studies to probe the active site structure of cholesterol oxidase

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. It has been used to clinically determine serum cholesterol level in the diagnosis of arteriosclerosis. It is also being developed for agricultural use as a pest control agent. Moreover, cholesterol oxidase is utilized in fundamental studies to characterize membrane structures.; It has been proposed that a hydrogen-bonding network comprised of the His447, Asn485, Glu361 and Wat541 helps to position the substrate relative to the FAD cofactor and coordinates general base and electrophilic catalysis for oxidation. A single mutant N485L was prepared to test this proposition. Kinetic analysis indicates that residue Asn485 plays a key role in catalysis of oxidation. Redox potential measurements for the FAD of wild type and N485L showed a 76 mV reduction in Em upon mutation. This reveals that the reason for the reduced catalytic activity of the N485L is the decrease in reduction potential of the FAD. This reduction is consistent with the loss of a π-cation-like stabilizing interaction between Asn485 and the cofactor.; The interplay of His447 and Glu361 was studied by constructing two mutants H447Q/E361Q and H447E/E361Q. Previous kinetic analysis suggested that Glu361 can compensate for the loss of histidine at position 447 by substituting as the general base catalyst for oxidation of cholesterol. Importantly, the construction of H447E/E361Q yielded an enzyme that is 31,000-fold slower than wild type in k_cat for oxidation. This mutant was used as a control for membrane binding effects in assays with caveolae membrane fractions; the native enzyme uncoupled platelet-derived growth factor receptor β (PDGFR β) autophosphorylation from tyrosine phosphorylation of neighboring proteins, and the H447E/E361Q mutant did not.; 2α,3α-cyclopropano-5α-cholestan-3β-ol inhibited cholesterol oxidase irreversibly and it will be useful for determining a cholesterol-bound enzyme structure by X-ray crystallography. However, the inactivated enzyme aggregates in the absence of detergent. To address this difficulty, detergents compatible with X-ray crystallography were screened and nonyl-β-D-glucoside was found to be the optimal detergent for preparation of inactivated enzyme. Large quantities of the inactivated-complex solution have been prepared for X-ray crystallization studies.