Separate genes encode the human type 1 (placenta,
breast tumors, other peripheral tissues) and type 2 (gonad, adrenal)
isoforms of
3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD1, 3 beta-HSD2). Mutagenesis of 3 beta-HSD1 produced the Y154F, H156Y and K158Q mutant
enzymes in the probable Y(154)-P-H(156)-S-K(158) catalytic motif. The H(156)Y mutant of the 3 beta-HSD1 created a chimera of the 3 beta-HSD2 motif (Y(154)-P-Y(156)-S-K(158)) in 3 beta-HSD1. The D241N, D257L, D258L and D265N mutants are in the potential
isomerase site of the 3 beta-HSD1
enzyme. Homology modeling with UDP-galactose-4-epimerase predicted that Asp(36) in the Rossmann-fold domain is responsible for the
NAD(H) specificity of human 3 beta-HSD1, and our D36A/K37R mutant tested that assignment. The H(156)Y mutant of the 3 beta-HSD1
enzyme shifted the substrate (
DHEA) kinetics to the 14-fold higher K(m) value measured for the 3 beta-HSD2 activity. From Dixon analysis,
epostane inhibited the 3 beta-HSD1 activity with 17-fold greater affinity compared to 3 beta-HSD2 and H(156)Y. The mutants of Tyr(154) and Lys(158) exhibited no
dehydrogenase activity and appear to be catalytic 3 beta-HSD residues. The D257L and D258L mutations eliminated
isomerase activity, suggesting that Asp(257) or Asp(258) may be catalytic residues for
isomerase activity. The D36A/K37R mutant shifted the cofactor preference of both 3 beta-HSD and
isomerase from
NAD(H) to
NADP(H). In addition to characterizing catalytic residues, these studies have identified the structural basis (His(156)) for an exploitable difference in the substrate and inhibition kinetics of 3 beta-HSD1 and 3 beta-HSD2. Hence, it may be possible to selectively inhibit human 3 beta-HSD1 to slow the growth of
hormone-sensitive
breast tumor cells and control placental steroidogenesis near term to prevent
premature labor.