THE COMMON I172N MUTATION CAUSES CONFORMATIONAL CHANGE OF CYTOCHROME P450C21 REVEALED BY SYSTEMATIC MUTATION, KINETIC, AND STRUCTURAL STUDIES

We have investigated the structure and function of P450c21 with regard to a conserved site around Ile-172 by site-directed mutagenesis makin g single amino acid substitutions of residues 169-173, Substitutions o f Ile-171 and -172 resulted in production of mutant proteins with dram atic reductions in enzymatic activities, indicating the importance of these two residues in maintaining the structure and function of P450c2 1, The I171N protein was present at a slightly lower level, due to a d ecreased rate of protein synthesis, The I172N apoprotein was synthesiz ed at the normal rate, but its heme-bound P450 form was present at a m uch lower level, This I172N protein was tightly integrated into the me mbrane of endoplasmic reticulum, similar to the wild type P450c21, as shown by immunofluorescence detection, alkaline extraction, and cellul ar fractionation, Kinetic studies indicated that I172N had a lower V,, , value, In addition, the I172N protein was more sensitive to proteina se K digestion, indicating a possible alteration of conformation, This conformational change may result in the lower yield of the I172N hemo protein and the reduced catalytic activity.