Structure-function studies of human 5-alpha reductase type 2 using site directed mutagenesis

Site directed mutagenesis of human steroid 5 alpha -reductase types 1 (5AR1 ) and 2 (5AR2) has been used to identify residues involved in inhibitor/sub strate binding by 5AR2. Replacing residues 21-24 (GALA) in 5AR2 with the an alogous residues 26-29 (AVFA) from 5AR1 did not significantly alter either the Km For testosterone or the Ki for the competitive inhibitor Finasteride . Replacement of AVFA in 5AR1 with GALA from 5AR2 however, significantly de creased the Km and increased the resistance to Finasteride. These findings confirm that 5AR1 residues 26-29 are involved in inhibitor/substrate bindin g but suggest residues 21-24 of 5AR2 are not. Replacing residues 20-29 (QCA VGCAVFA) of 5AR1 with the analogous residues 15-24 (ATLVAL-GALA) from 5AR2, changed the Km and Ki to values approaching those for wild type 5AR2. Repl acing residues VAL in wild type 5AR2 with VGC from 5AR1 did not change Km o r Ki but replacing ATL in 5AR2 with QCA from 5AR1 significantly decreased t he Km and increased the resistance to Finasteride. Conversely, replacing QC A with ATL in 5AR1 containing GALA in place of AVFA, increased the Km and d ecreased resistance to Finasteride. These findings indicate residues 15-17 of human 5AR2 participate in inhibitor/substrate binding whereas residues 1 8-20 do not. (C) 2001 Elsevier Science Ltd. All rights reserved.