Substrate-Dependent Allosteric Regulation in Cytochrome P450cam (CYP101A1).

Various biophysical methods have provided evidence of a second substrate binding site in the well-studied cytochrome P450cam, although the location and biological relevance of this site has remained elusive. A related question is how substrate and product binding and egress occurs. While many active site access channels have been hypothesized, only one, channel 1, has been experimentally validated. In this study, molecular dynamics simulations reveal an allosteric site related to substrate binding and product egress. The remote allosteric site opens channel 1 and primes the formation of a new channel that is roughly perpendicular to channel 1. Substrate entry to the active site via channel 1 as well as substrate/product egress via channel 2 is observed after binding of a second molecule of substrate to the allosteric site, indicating cooperativity between these two sites. These results are consistent with and bring together many early and recent experimental results to reveal a dynamic interplay between a weak allosteric site and substrate binding to the active site that controls P450cam activity.