Stereoselective hydroxylation of norcamphor by cytochrome P450cam. Experimental verification of molecular dynamics simulations
| Autor: | P J, Loida, S G, Sligar, M D, Paulsen, G E, Arnold, R L, Ornstein |
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| Rok vydání: | 1995 |
| Předmět: |
Models
Molecular Binding Sites Camphor 5-Monooxygenase Molecular Structure Protein Conformation Stereoisomerism Hydroxylation NAD Norbornanes Recombinant Proteins Mixed Function Oxygenases Substrate Specificity Kinetics Cytochrome P-450 Enzyme System Escherichia coli Computer Simulation Oxidation-Reduction |
| Zdroj: | The Journal of biological chemistry. 270(10) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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