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The active site

The substrate, (1R)-camphor, binds in the active site as shown with its keto oxygen accepting a hydrogen bond from Tyr 96. It is hydroxylated at the 5-exo position by cytochrome P450cam.
Comparative analysis of the binding properties of the (1R)- and (1S)-camphor enantiomers highlight the subtle level at which cytochrome P450cam is optimized for its natural substrate. Data from static and time-resolved FTIR spectroscopy to monitor the CO stretch band[1,10] , 13C NMR [6], temperature-jump and pressure-jump analyses, x-ray crystallography and molecular dynamics simulations show that (1S)-camphor is more mobile in the active site and that the active site is slightly more hydrated than in the case of (1R)-camphor [10]. These differences may account for the greater uncoupling for (1S)-camphor than (1R)-camphor. They also point to the complementarity in the van der Waals interactions between (1R)-camphor and the protein.
Nevertheless, there is an interfacial cavity adjacent to the camphor keto oxygen large enough to amply accommodate a water molecule[11] although molecular dynamics calculations [4] show that it is a predominantly unhydrated cavity and no water molecules are assigned in this position in the protein crystal structure. Substrate analogues were designed to fill this cavity in the expectation that such analogues would bind with greater affinity than camphor[5]. Consistent with the modelling, a compound with a 3-carbon substituent, endo-borneol allyl ether, was found to bind best with a kd=0.6 +- 0.1 microM (cf camphor with kd=1.7 +- 0.2 microM). Binding of this camphor analogue to the Y96F mutant was much enhanced over the binding of camphor (kd=0.2 microM cf camphor kd=2.2 microM) indicating that hydrogen bonding plays a less important role in its binding. Binding enthalpies calculated from the simulations, taking all solvent contributions into account, agree very well with experimental binding enthalpies. They are not correlated with binding affinities as binding of the substrate analogues is characterized by enthalpy-entropy compensation. Fe spin equilibrium and hydrostatic pressure measurements indicate that endo-borneol allyl ether displaces about the same amount of water from the active site as camphor. However, recent T-jump measurements of protein relaxation (Schulze and Jung, unpublished data) and crystallographic data (Schlichting, unpublished data) indicate that this compound has greater mobility in the active site and this may be the reason for its reduced catalytic efficiency (Kozin and Hui Bon Hoa, unpublished data) compared to camphor.

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Wade et al. (1996) Fundamentals of Enzyme-Ligand Interactions in Cytochrome P450cam
POPE5 Conference Proceedings
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