IMPROVED BINDING OF CYTOCHROME P450CAM SUBSTRATE-ANALOGS DESIGNED TO FILL EXTRA SPACE IN THE SUBSTRATE-BINDING POCKET

Cytochrome P450cam catalyzes the 5-exo-hydroxylation of camphor. Camph or analogues were designed to fill an empty region of the substrate bi nding pocket with the expectation that they would bind more tightly th an camphor itself due to increased van der Waals interactions with the protein and the displacement of any solvent occupying this site. A se ries of compounds (endo-borneol methyl ether endo-borneol propyl ether , endo-borneol allyl ether and endo-borneol dimethyl allyl ether) were synthesized with substituents at the camphor carbonyl oxygen. The spi n conversion and thermodynamic properties of this series of compounds were measured for wild type and Y96F mutant cytochrome P450cam and wer e interpreted in the context of molecular dynamics simulations of the camphor analogues in the P450 binding site and in solution. Compounds with a 3-carbon chain substituent were predicted to match the size of the unoccupied region most optimally and thus bind best. Consistent wi th this prediction, the borneol allyl ether binds to cytochrome P450ca m with highest affinity with a K-d = 0.6 +/- 0.1 mu M (compared to a K -d = 1.7 +/- 0.2 mu M for camphor under the same experimental conditio ns). Binding of the camphor analogues to the Y96F mutant is much enhan ced over the binding of camphor, indicating that hydrogen bonding play s a less important role in binding of these analogues. Binding enthalp ies calculated from the simulations, taking all solvent contributions into account, agree very well with experimental binding enthalpies. Bi nding affinity is not however correlated with the calculated binding e nthalpy because the binding of the substrate analogues is characterize d by enthalpy-entropy compensation. The new compounds are useful probe s for further studies of the mechanism of cytochrome P450cam due to th eir high binding affinities and high spin properties.