Stereoselective Hydroxylation of Norcamphor by Cytochrome P450cam
EXPERIMENTAL VERIFICATION OF MOLECULAR DYNAMICS SIMULATIONS (*)
- From the (1)School of Chemical Sciences and The Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana, Illinois 61801 (2)Environmental Molecular Sciences Laboratory, Pacific Northwest Laboratory, Richland, Washington 99352
- ↵§ Authors to whom correspondence should be addressed.
Abstract
The stereoselectivity of cytochrome P450cam hydroxylation has been investigated with the enantiomerically pure substrate analog norcamphor. (1R)- and (1S)-norcamphor (>92 enantiomeric excess) were characterized in the hydroxylation reaction with cytochrome P450cam with respect to the product profile, steady state kinetics, coupling efficiency, and free energy of substrate dissociation. The experimental results demonstrate regiospecificity that is enantiomer-specific and confirm our previously reported prediction that (1R)-norcamphor is hydroxylated preferentially at the 5-carbon and (1S)-norcamphor at the 6-carbon (Bass, M. B., and Ornstein, R. L.(1993) J. Comput. Chem. 14, 541-548); these simulation results are now compared with simulations involving a ferryl oxygen intermediate. Hydroxylation of (1R)-norcamphor was found at the 5-, 6-, and 3-carbons in a ratio of 65:30:5 (respectively), whereas (1S)-norcamphor was oxidized to produce a 28:62:10 ratio of the same products. With the exception of the regiospecificity, all of the reaction and physical parameters are similar for each enantiomer of norcamphor. These results show that the position of the carbonyl group on the hydrocarbon skeleton of norcamphor plays a role in determining the average orientation of this substrate in the active site and suggests that hydrogen bonding interactions can aid in directing the regiospecificity and stereospecificity of the hydroxylation reaction catalyzed by cytochrome P450cam.
Footnotes
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↵* This work was supported by Grant GM 33775 from the National Institutes of Health and Grant KP0402 from the Health Effects and Life Science Research Division of the Office of Health and Environmental Research of the Department of Energy (to R. L. O.). Pacific Northwest Laboratory is operated for the United States Department of Energy by Battelle Memorial Institute under Contract DE-AC06-76RLO-1830. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received August 18, 1994.
- Revision received October 18, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
