MOLECULAR-DYNAMICS SIMULATIONS INDICATE THAT F87W,T185F-CYTOCHROME P450CAM MAY REDUCTIVELY DEHALOGENATE 1,1,1-TRICHLOROETHANE

Cytochrome P450cam is capable of reductively dehalogenating several ch lorinated alkanes at low, but measurable, rates. In previous investiga tions of structure-function relationships in this enzyme using molecul ar dynamics simulations, we noticed that 1,1,1-trichloroethane (TCA) e xhibits a very high degree of mobility in the active site due to its s maller molecular volume relative to the native substrate, camphor(1,2) . Several amino acid sidechains lining the active site also exhibit si gnificant dynamic fluctuations, possibly as a result of poor steric co mplementarity to TCA. Guided by these results, we modeled double (F87W , T185F) and triple (F87W, T185E V295I) mutants of P450cam, which prov ide additional bulk in the active site and increase the frequency of h eme-substrate collision. Molecular dynamics simulations (300 ps on eac h protein) indicate that these mutants do not significantly perturb th e three-dimensional fold of the enzyme, or local structure in the regi on of the active site. Both mutants bind the substrate more stably nea r the heme than the wild-type. Interestingly, however, the bulkier tri ple mutant seems to actually inhibit heme-substrate interactions relat ive to the double mutant. Over the final 200 ps of simulation, TCA is within 1 Angstrom of nonbonded contact with the heme 25% more often in the double mutant versus the wild-type. The triple mutant, on the oth er hand, binds TCA within 1 Angstrom of the heme only 15% as often as the wild-type. These results indicate that the double mutant may reduc tively dehalogenate TCA, a property not observed for the native protei n. Implications for other experimentally measurable parameters are dis cussed.