Substrate routes to the buried active site may vary among cytochromes P450: mutagenesis of the F-G region in P450 2B1.

Until recently, all known structures of bacterial cytochromes P450 suggested that substrate access to the buried active site occurred via the F-G region, a surface loop distal to the heme cavity. However, the structure of P450 51 indicates a large opening from the protein surface along the I helix N-terminus, at right angles to the F-G channel. The single available microsomal P450 structure (2C5) does not obviously favor one potential access route over the other. To determine whether the F-G region forms part of the substrate access channel in the microsomal cytochrome P450 2B1, 11 residues between positions 208 and 230 were substituted with smaller and larger side chains in a highly expressed truncated form of the enzyme. Steady-state kinetic parameters were determined with the substrates testosterone, 7-ethoxy-4-trifluoromethylcoumarin (7-EFC), and 7-benzyoxyresorufin (7-BR). The largest changes, 2-6-fold increases in k(cat) with testosterone and 7-EFC, were observed for L209A, which also exhibits an altered testosterone metabolite profile and probably forms part of the active site roof. F219W demonstrated little or no activity with any of the three substrates examined, although the K(s) value for benzphetamine binding was unaltered. S221F showed little activity with 7-BR. No significant changes were observed in K(m)(testosterone) or S(50)(7-EFC) values for any of the mutants, in stark contrast to the 10-fold and 100-fold changes in K(m) observed for mutants in this region of other cytochromes P450. The minimal changes in 2B1 do not support access via the F-G region of 2B1 and suggest the alternate access route identified in P450 51.