Dynamics Investigation of the Cytochrome P450cam Active Site Mutant Thr252ALA

Abstract

Cytochrome P450s are vital heme-containing enzymes that facilitate catalysis in a wide variety of biochemical processes including carcinogenesis, drug metabolism, steroid biosynthesis and toxin degradation. Because of its ability to catalyze the hydroxylation mechanism of camphor in its active site, Cytochrome P450cam served as the model system for studying P450s. The crystal structure of a P450cam mutant, in which the active site Threonine252 (Thr252) has been replaced with an alanine (ala) residue (Thr252Ala), was solved at 2.2Å resolution. According to sequence alignment analysis, Thr252 is highly conserved among P450s, and it was suggested to be an essential active site residue, forming part of the dioxygen-binding site. Mutation of the active site Thr to Ala changes local conformation of the active site residues and affects hydroxylation of the substrate, producing hydrogen peroxide and “excess” water instead of the substrate polarizing product, 5-exo-hydroxycamphor. In the mutant active site, a solvent not present in the native enzyme is positioned in the dioxygen-binding region and the network of hydrogen bonds is restructured accordingly. These factors are believed to be critical to enzymatic reaction changes of P450cam mutant from the wild-type, most notably the proton delivery. Molecular dynamics (MD) simulation has been performed on the mutant to predict the relevant structural dynamics, especially those of the active site residues. Neutron scattering (NS) spectra calculations have been extrapolated from the simulation and compared with the results with the indicated crystallographic findings and those of the wild-type P450cam simulations already empirically verified via experimental neutron scattering. Preliminary results predict that local interactions around the heme and cam site have been altered midway through the trajectory. The preliminary data has also provided insight to understanding the local water diffusion dynamics of the CYP450cam mutant active site.