SUBSTRATE MOBILITY IN A DEEPLY BURIED ACTIVE-SITE - ANALYSIS OF NORCAMPHOR BOUND TO CYTOCHROME-P-450(CAM) AS DETERMINED BY A 201-PSEC MOLECULAR-DYNAMICS SIMULATION

While cytochrome P-450(cam) catalyzes the hydroxylation of camphor to 5-exo-hydroxycamphor with 100% stereospecificity, norcamphor is hydroxylated by this enzyme yielding 45% 5-exo-, 47% 6-exo-, and 8% 3-exo-hydroxynorcamphor (Atkins, W.M., Sligar, S.G., J. Am. Chem. Soc. 109:3754-3760, 1987). The present study describes a 201-psec molecular dynamics (MD) simulation of norcamphor-bound cytochrome P-450cam to elucidate the relationship between substrate conformational mobility and formation of alternative products. First, these data suggest that the product specificity is, at least partially, due to the mobility of the substrate within the active site. Second, the high mobility of norcamphor in the active site leads to an average increase in separation between the heme iron and the substrate of about 1.0 angstrom; this increase in separation may be the cause of the uncoupling of electron transfer when norcamphor is the substrate. Third, the active site water located in the norcamphor-bound crystal structure possesses mobility that correlates well with the spin-state equilibrium of this enzyme-substrate complex.