PubMed1995
Cytochrome P450cam is capable of reductively dehalogenating several chlorinated alkanes at low, but measurable, rates. In previous investigations of structure-function relationships in this enzyme using molecular dynamics simulations, we noticed that 1,1,1-trichloroethane (TCA) exhibits a very high degree of mobility in the active site due to its smaller molecular volume relative to the native substrate, camphor(1,2). Several amino acid sidechains lining the active site also exhibit significant dynamic fluctuations, possibly as a result of poor steric complementary to TCA. Guided by these results, we modeled double (F87W, T185F) and triple (F87W, T185F, V295I) mutants of P450cam, which provide additional bulk in the active site and increase the frequency of heme-substrate collision. Molecular dynamics simulations (300 ps on each protein) indicate that these mutants do not significantly perturb the three-dimensional fold of the enzyme, or local structure in the region of the active site. Both mutants bind the substrate more stably near the heme than the wild-type. Interestingly, however, the bulkier triple mutant seems to actually inhibit heme-substrate interactions relative to the double mutant. Over the final 200 ps of simulation, TCA is within 1 A of nonbonded contact with the heme 25% more often in the double mutant versus the wild-type. The triple mutant, on the other hand, binds TCA within 1 A of the heme only 15% as often as the wild-type. These results indicate that the double mutant may reductively dehalogenate TCA, a property not observed for the native protein. Implications for other experimentally measurable parameters are discussed.