Site-directed mutagenesis studies of the NADPH-binding domain of rat steroid 5alpha-reductase (isozyme-1) I: analysis of aromatic and hydroxylated amino acid residues.
Previous studies have shown that the reduced nicotinamide adenine dinucleotide phosphate (NADPH)- binding domain of rat liver microsomal steroid 5alpha-reductase isozyme-1 (r5alphaR-1) is in a highly conserved region of the polypeptide sequence (residues 160-190). In this study, we investigated, by site-directed mutagenesis, the role of hydroxylated and aromatic amino acids within the NADPH-binding domain. The r5alphaR-1 cDNA was cloned into a pCMV vector, and the double strand site-directed mutagenesis method was used to create mutants Y179F, Y179S, Y189F, Y189S, S164A, S164T, and Y187F, which were subsequently expressed in COS-1 cells. Kinetic studies of the expressed enzymes showed that the mutation Y179F resulted in an approximately 40-fold increase in the Km for NADPH versus wild-type, with only a 2-fold increase in the Km for testosterone. The mutants Y189F and S164A showed smaller increases (4 and 6-fold) in Kms for NADPH and no significant change in the Km for testosterone, whereas Y189S had kinetic properties similar to the wild-type r5alphaR-1. Mutants Y179S and S164T both resulted in inactive enzymes, whereas F187Y showed an approximately 5-fold decrease in Km for NADPH and a significant increase (approximately 18-fold) in the Km for testosterone. The results suggest that the -OH functionality of Y179 is involved in cofactor binding, but is not essential for the activity of the enzyme, whereas the -OH functionalities of Y189 and S164 play lesser roles in cofactor binding to r5alphaR-1 and may not be required for enzyme activity. On the other hand, the residue F187 may be important for the binding of both NADPH and testosterone.
VAMC and Department of OB/GYN, University of Kentucky, Lexington, USA.
M
M
Wang
A K
AK
Bhattacharyya
M F
MF
Taylor
H H
HH
Tai
D C
DC
Collins
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D006900:Hydroxylation; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism*; D017354:Point Mutation; D051381:Rats; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D000596:Amino Acids; D007527:Isoenzymes; D009249:NADP; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase