| Abstract
| - Cytochrome P450cam catalyzes the 5-exo-hydroxylation ofcamphor. Camphor analogueswere designed to fill an empty region of the substrate binding pocketwith the expectation that they wouldbind more tightly than camphor itself due to increased van der Waalsinteractions with the protein and thedisplacement of any solvent occupying this site. A series ofcompounds (endo-borneol methyl ether,endo-borneol propyl ether, endo-borneol allylether and endo-borneol dimethyl allyl ether) weresynthesizedwith substituents at the camphor carbonyl oxygen. The spinconversion and thermodynamic propertiesof this series of compounds were measured for wild type and Y96F mutantcytochrome P450cam andwere interpreted in the context of molecular dynamics simulations ofthe camphor analogues in the P450binding site and in solution. Compounds with a 3-carbon chainsubstituent were predicted to match thesize of the unoccupied region most optimally and thus bind best.Consistent with this prediction, theborneol allyl ether binds to cytochrome P450cam with highest affinitywith a Kd = 0.6 ± 0.1 μM(comparedto a Kd = 1.7 ± 0.2 μM for camphor underthe same experimental conditions). Binding of thecamphoranalogues to the Y96F mutant is much enhanced over the binding ofcamphor, indicating that hydrogenbonding plays a less important role in binding of these analogues.Binding enthalpies calculated fromthe simulations, taking all solvent contributions into account, agreevery well with experimental bindingenthalpies. Binding affinity is not however correlated with thecalculated binding enthalpy because thebinding of the substrate analogues is characterized byenthalpy−entropy compensation. The newcompounds are useful probes for further studies of the mechanism ofcytochrome P450cam due to theirhigh binding affinities and high spin properties.
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