STEREOSELECTIVE HYDROXYLATION OF NORCAMPHOR BY CYTOCHROME P450(CAM) -EXPERIMENTAL-VERIFICATION OF MOLECULAR-DYNAMICS SIMULATIONS

The stereoselectivity of cytochrome P450(cam) hydroxylation has been i nvestigated with the enantiomerically pure substrate analog norcamphor , (1R)- and (1S)-norcamphor (>92 enantiomeric excess) were characteriz ed in the hydroxylation reaction with cytochrome P450(cam) with respec t to the product profile, steady state kinetics, coupling efficiency, and free energy of substrate dissociation. The experimental results de monstrate regiospecificity that is enantiomer-specific and confirm our previously reported prediction that (1R)-norcamphor is hydroxylated p referentially at the 5-carbon and (1S) norcamphor at the 6 carbon (Bas s, M. B., and Ornstein, R, L, (1993) J. Comput. Chem, 14, 541-548); th ese simulation results are now compared with simulations involving a f erryl oxygen intermediate. Hydroxylation of (1R)-norcamphor was found at the 5, 6-, and S-carbons in a ratio of 65:30:5 (respectively), wher eas (1S)-norcamphor was oxidized to produce a 28:62:10 ratio of the sa me products. With the exception of the re giospecificity, all of the r eaction and physical parameters are similar for each enantiomer of nor camphor. These results show that the position of the carbonyl group on the hydrocarbon skeleton of norcamphor plays a role in determining th e average orientation of this substrate in the active site and suggest s that hydrogen bonding interactions can aid in directing the regiospe cificity and stereospecificity of the hydroxylation reaction catalyzed by cytochrome P450(cam).