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 ,
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
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
Nevertheless, there is an interfacial cavity
adjacent to the camphor keto oxygen large
enough to amply accommodate a water molecule although molecular dynamics
calculations  show that it is a predominantly unhydrated cavity and no
water molecules are assigned in this position in the protein crystal structure.
were designed to fill this cavity in the expectation that
such analogues would bind with greater affinity than camphor.
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
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unpublished data) and crystallographic data (Schlichting, unpublished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and Hui Bon Hoa, unpublished data) compared to camphor.