Enzymologic Defect in the R347H and R358Q Mutations in P450c17 that Cause Selective 17,20-Lyase Deficiency • 425

P450c17, the qualitative regulator of steroid hormone biosynthesis, possesses both 17α-hydroxylase and 17,20-lyase activities. We previously reported that two patients with isolated 17,20-lyase deficiency carried the P450c17 mutations R347H and R358Q. Transfection of COS-1 cells showed that each mutation retains greater than 60% of the 17α-hydroxylase activity but negligible lyase activity, and our computer modeling indicated that these two affected residues lie in the electron donor binding region of P450c17(Nature Genet. 17:201, 1997). We have now studied the enzymatic activity of these two mutants in transfected COS-1 cells and in microsomes from genetically engineered yeast. In transfections with COS-1 cells, co-expression of R347H and the requisite NADPH-oxidoreductase (OR) increases lyase activity 18-fold; triple transfections with both OR and cytochrome b₅ enhances lyase activity nearly 50-fold. Co-expressed OR more modestly augments R358Q lyase activity by 6-fold; in the presence of both OR and cytochrome b₅, the lyase activity increases 15-fold. Assays of the mutant P450c17 activity in yeast microsomes demonstrate that co-expression of the requisite NADPH-oxidoreductase (OR) decreases the apparent K_m of the R347H mutant for pregnenolone from 0.48μM to 0.24μM and from 0.54μM to 0.29μM for the R358Q mutant. In both transfected COS-1 cells and in isolated yeast microsomes, cytochrome b₅ restores a portion of the missing lyase activity, but its effect is most significant in the presence of co-expressed OR. In the yeast system, the molar ratio of cytochrome b₅:P450 needed to produce significant lyase activity for each mutant is 10-fold greater than the optimal ratio for wild-type P450c17. Thus the selective loss of 17,20-lyase activity could be partially restored by optimizing the electron-transfer system. This lends further credence to our proposal that these two mutations disrupt residues involved in proper coupling of P450c17 with its electron donors, and further supports our hypothesis that the differential regulation of the 17α-hydroxylase and 17,20-lyase activities of P450c17 is at the level of electron transfer.