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Proposed Substrate Access Channel

The crystal structure of cytochrome P450cam shows no channel between the buried active site and the protein surface large enough to permit passage of camphor. However there is a small hydrophobic opening as shown by the arrow and this was suggested to be a substrate access channel by Poulos [8,9] that, with protein motion could expand to allow passage of camphor. More recently, the structure of cytochrome P450-BM3 has been solved [3] and shows a large channel connecting solvent and the binding site in the position shown by the arrow.

The binding of camphor to cytochrome P450cam can be considered a two-step process: diffusion of camphor to the active site followed by a rearrangment in the active site accompanied by a low-spin to high-spin transition. Experiments show that the equilibrium constant for the first step is dependent on ionic strength and solvent dielectric constant [2] i.e. that electrostatic interactions are important in this step. The ionic strength and solvent dielectric dependence is much reduced for the D251N mutant indicating the importance of this residue for the binding of camphor. This residue participates in a tetrad of salt-links adjacent to the proposed access channel. Electrostatics calculations of the stability of all the salt-links in cytochrome P450cam show the salt-links to Asp 251 to be unusually stable indicating that the enzyme may have evolved these salt-links to regulate substrate access.

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