Substrate specificity of cytochrome P450cam for L ‐ and D ‐ norcamphor as studied by molecular dynamics simulations

In our earlier molecular dynamics simulations, we found that there was a discrepancy between the predicted and experimental product ratios when norcamphor is hydroxylated by cytochrome P450cam. The experimental results suggest that there is a nearly equimolar ratio between the 5‐ and 6‐hydroxynorcamphor (45% 5‐, 47% 6‐, and 8% 3‐hydroxynorcamphor) [W.M. Atkins and S.J. Sligar, J. Am. Chem. Soc. , 109 , 3754 (1987)]. Our previous simulations predicted predominately from 68–88% 5‐hydroxynorcamphor [M.B. Bass et al., Prot. Struct. Funct. Genet. , 13 , 26 (1992); M.B. Bass et al., Proc. Natl. Acad. Sci. U.S.A. , submitted]. One possible explanation for this discrepancy is that the simulations were performed using D ‐norcamphor while the experiments were conducted with racemic norcamphor. The suggestion that norcamphor is the D ‐isomer was based upon the similarity with the native substrate D ‐camphor. Indeed, the reported crystallographic structure for norcamphor‐bound P450cam models norcamphor as the D ‐isomer. Unfortunately, the two stereomers have never been separated. The simulations presented here model the L ‐isomer of norcamphor. Three simulations each of the L ‐ and D ‐isomers of norcamphor bound to cytochrome P450cam were compared to account for the effects due to substrate orientation and the assignment of random velocities. The results presented here show that the L ‐isomer of norcamphor is predicted to give rise to predominately 6‐hydroxynorcamphor, while the D ‐isomer gives rise to mainly 5‐hydroxynorcamphor. From this data, we infer that racemic norcamphor will give rise to nonracemic 5‐ and 6‐hydroxynorcamphors after oxidation by cytochrome P450cam. © 1993 John Wiley & Sons, Inc.