Functional analysis of the linker region of P450 2C2

The linker region connecting the N-tenninal signal anchor and ihr cyloplas mir catalytic domain of P4r)0 2C2 contains I wo segments with distinct sequent e properties: a (ily-rirh and a Pro-rich region. In the Giy-rich region, substitution of the (ilv residues with Ala or Pro or the sequence from 22 to 2K with 7 Ala did not reduce laurate hydroxylase activity of the proteins expressed in COS-1 cells. Deletion of residues 22-28 or substitution with 7 Vil or 2 Ala inactivated the protein. Activity increased progressively with substitution of il or -1 Ala to activities similar to wild-type. Correspondingly, lengthening the linker from 2 to 7 Ala resulted in a progressive increase in P450 and a decrease in inactive P 120 for the mutants expressed in bacteria or insect cells. Subati tution of 7 Val resulted in only P420 and deletion of 22-2 resulted in neither IM50 nor P420. The activities per nrnole P450 were similar for wild type and the muUnts with 2 to 7 Ala substituted. These data are consistent with a role for the (lly-rich region as a linker which facilitates the folding of P-150 into a functional protein, but is not required for the activity of the folded protein. Similarly, mutations at Pro30 and Pro33 in the Pro-rich region which reduce activity in COS 1 cells, also resulted in reduced IM 50 and increased 1420 for the mu'ants expressed in bacteria or insect cells. Fliese data suggest the Pro rich region is also critical for the folding of P-150.