Serine 124 completes the Tyr, Lys and Ser triad responsible for the catalysis of human type 1 3beta-hydroxysteroid dehydrogenase.
Human 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a key steroidogenic enzyme that catalyzes the first step in the conversion of circulating dehydroepiandrosterone (DHEA), pregnenolone or 17alpha-hydroxypregenolone to produce the appropriate, active steroid hormone(s): estradiol, testosterone, progesterone, aldosterone or cortisol respectively. Our mutagenesis studies have identified Tyr154 and Lys158 as catalytic residues for the 3beta-HSD reaction. Our three-dimensional homology model of 3beta-HSD shows that Tyr154 and Lys158 are oriented near the 3beta-hydroxyl group of the bound substrate steroid, and predicts that Ser123 or Ser124 completes a Tyr-Lys-Ser catalytic triad that operates in many other dehydrogenases. The S123A and S124A mutants of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) were created by PCR-based mutagenesis, expressed in insect cells using baculovirus and purified to homogeneity. The S124A mutant exhibits no 3beta-HSD activity and has a K(m) value (83.6 microM) for the isomerase substrate that is threefold greater than that of wild-type 1 isomerase. In contrast, S123A has substantial 3beta-HSD activity (DHEA K(m)=11.2 microM; k(cat)=0.8 min(-1)) and utilizes isomerase substrate, 5-androstene-3,17-dione, with a K(m) value (27.6 microM) that is almost identical to wild-type. The K(m) value (4.3 microM) of S124A for NADH as an allosteric activator of isomerase is similar to that of the wild-type 1 enzyme, indicating that Ser124 is not involved in cofactor binding. S123A utilizes NAD as a cofactor for 3beta-HSD and NADH as the activator for isomerase with K(m) values that are similar to wild-type. The 3beta-HSD activities of S123A and wild-type 3beta-HSD increase by 2.7-fold when the pH is raised from 7.4 to the optimal pH 9.7, but S124A exhibits very low residual 3beta-HSD activity that is pH-independent. These kinetic analyses strongly suggest that the Ser124 residue completes the catalytic triad for the 3beta-HSD activity. Since there are 29 Ser residues in the primary structure of human 3beta-HSD1, our homology model of the catalytic domain has been validated by this accurate prediction. A role for Ser124 in the binding of the isomerase substrate, which is the 3beta-HSD product-steroid of the bifunctional enzyme protein, is also suggested. These observations further characterize the structure/function relationships of human 3beta-HSD and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta to control the timing of labor or in hormone-sensitive breast tumors to slow their growth.
Division of Basic Medical Sciences and Department of Obstetrics-Gynecology, Mercer University School of Medicine, Macon, Georgia 31207, USA. Thomas_J@mercer.edu
J L
JL
Thomas
W L
WL
Duax
A
A
Addlagatta
L A
LA
Scaccia
K A
KA
Frizzell
S B
SB
Carloni
D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D012694:Serine / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D017931:DNA Primers; D014443:Tyrosine; D012694:Serine; D015096:3-Hydroxysteroid Dehydrogenases; D008239:Lysine