BIOL 186 |
| Cholesterol oxidases catalyze the oxidation and isomerization of cholesterol to cholest-4-en-3-one with concomitant reduction of an FAD cofactor, which is regenerated by oxygen. The atomic crystal structure of cholesterol oxidase reveals a hydrophobic tunnel that extends from the active site cavity to the surface of the protein. This tunnel may serve to direct access of oxygen to the active site. Mutations were designed to block passage through the tunnel by steric hindrance. The mutated tunnel residues are 10-15 Å from the FAD cofactor and their mutation reduces the rate of oxidation 30-60 fold while the rate of isomerization is almost unchanged. Deuterium substitution at the 3α-position of cholesterol does not affect the rate of oxidation. Steady-state two substrate kinetic analysis shows that the apparent binding affinity of the mutant enzymes for molecular oxygen has decreased at least 10-fold in the mutants. |
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Enzymes
4:30 PM-6:30 PM, Wednesday, 13 September 2006 Moscone Center -- Hall D, Poster
Sci-Mix
Division of Biological Chemistry |