| The catalytic mechanism of cholesterol oxidase has been extensively studied, but the steps involving the usage of molecular oxygen are still not fully understood. Recently, a crystal structure of cholesterol oxidase was solved at sub-Angstrom resolution (Lario PI, Sampson N and Vrielink A, J Mol. Biol. 2003; 326:1635-50). Multiple side-chain conformations were observed for the active site residues. The conformations of Asn485 suggested that it acts as a gate to modulate oxygen entry through a tunnel. The tunnel residues adopt multiple conformations as well. In this work, validation of the catalytic function for other tunnels was sought. Mutations of tunnel residues result in 30-60 fold reductions in the rate of oxidation, but the rate of isomerization is almost unchanged. Hydride transfer from sterol to flavin adenine dinucleotide (FAD) is no longer rate limiting in the tunnel mutants. The wild-type enzyme forms a ternary complex with both substrates, cholesterol and oxygen. The mutant enzymes are kinetically cooperative with respect to oxygen. A mnemonic model was constructed to explain the observed kinetic cooperativity. As predicted by the model, The mutant reactions are not cooperative with respect to cholesterol and kinetic cooperativity increases with increasing cholesterol concentration. Also consistent with the model, positive cooperativity is lost when a poor substrate reduces the rate of subsequent catalytic steps. The crystal structure of the mutant, F359W, supports the kinetic interpretation. |
