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1.
Mol Cell Endocrinol ; 215(1-2): 73-82, 2004 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-15026177

RESUMEN

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.


Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Mutación , NADP/metabolismo , NAD/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/genética , Secuencia de Aminoácidos , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad
2.
Endocr Res ; 28(4): 471-5, 2002 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12530651

RESUMEN

Two distinct genes encode the human type 1 (placenta, mammary gland) and type 2 (adrenal, gonad) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD). We have produced the Y154F, H156Y, and K158Q mutant enzymes in the Y154-P-H156-S-K158 motif of the human type 1 3beta-HSD/isomerase. The H156Y mutant was created to produce a chimera of the type 2 enzyme motif (Y154-P-Y156-S-K158) in the type 1 enzyme. The wild-type (WT) 1 and 2 plus the mutant enzymes were expressed and purified. The Km for dehydroepiandrosterone and Ki for epostane measured with both the H156Y mutant and WT 2 are 13-fold to 17-fold greater than those values obtained with the WT 1 3beta-HSD. The Y154F and K158Q mutants exhibit no 3beta-HSD but have significant isomerase activity. Thus, H156 in WT 1 vs. Y156 in WT 2 accounts for the substantially higher affinity of WT 1 3beta-HSD activity for these substrate and inhibitor steroids relative to the WT 2 enzyme.


Asunto(s)
Complejos Multienzimáticos/antagonistas & inhibidores , Progesterona Reductasa/antagonistas & inhibidores , Esteroide Isomerasas/antagonistas & inhibidores , Androstenoles/farmacología , Animales , Línea Celular , Deshidroepiandrosterona/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Insectos , Isoenzimas/antagonistas & inhibidores , Cinética , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mutación , Progesterona Reductasa/genética , Progesterona Reductasa/metabolismo , Esteroide Isomerasas/genética , Esteroide Isomerasas/metabolismo
3.
J Biol Chem ; 277(45): 42795-801, 2002 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-12205101

RESUMEN

Two distinct genes encode the 93% homologous type 1 (placenta, peripheral tissues) and type 2 (adrenals, gonads) 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) in humans. Mutagenesis studies using the type 1 enzyme have produced the Y154F and K158Q mutant enzymes in the Y(154)-P-H(156)-S-K(158) motif as well as the Y269S and K273Q mutants from a second motif, Y(269)-T-L-S-K(273), both of which are present in the primary structure of the human type 1 3beta-HSD/isomerase. In addition, the H156Y mutant of the type 1 enzyme has created a chimera of the type 2 enzyme motif (Y(154)-P-Y(156)-S-K(158)) in the type 1 enzyme. The mutant and wild-type enzymes have been expressed and purified. The K(m) value of dehydroepiandrosterone is 13-fold greater, and the maximal turnover rate (K(cat)) is 2-fold greater for wild-type 2 3beta-HSD compared with the wild-type 1 3beta-HSD activity. The H156Y mutant of the type 1 enzyme has substrate kinetic constants for 3beta-HSD activity that are very similar to those of the wild-type 2 enzyme. Dixon analysis shows that epostane inhibits the 3beta-HSD activity of the wild-type 1 enzyme with 14-17-fold greater affinity compared with the wild-type 2 and H156Y enzymes. The Y154F and K158Q mutants exhibit no 3beta-HSD activity, have substantial isomerase activity, and utilize substrate with K(m) values similar to those of wild-type 1 isomerase. The Y269S and K273Q mutants have low, pH-dependent 3beta-HSD activity, exhibit only 5% of the maximal isomerase activity, and utilize the isomerase substrate very poorly. From these studies, a structural basis for the profound differences in the substrate and inhibition kinetics of the wild-type 1 and 2 3beta-HSD, plus a catalytic role for the Tyr(154) and Lys(158) residues in the 3beta-HSD reaction have been identified. These advances in our understanding of the structure/function of human type 1 and 2 3beta-HSD/isomerase may lead to the design of selective inhibitors of the type 1 enzyme not only in placenta to control the onset of labor but also in hormone-sensitive breast, prostate, and choriocarcinoma tumors to slow their growth.


Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Catálisis , Cartilla de ADN , Humanos , Cinética , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mutagénesis Sitio-Dirigida , Progesterona Reductasa/química , Progesterona Reductasa/genética , Progesterona Reductasa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Esteroide Isomerasas/química , Esteroide Isomerasas/genética , Esteroide Isomerasas/metabolismo , Especificidad por Sustrato
4.
J Biol Chem ; 278(37): 35483-90, 2003 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-12832414

RESUMEN

Human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD/isomerase) catalyzes the two sequential enzyme reactions on a single protein that converts dehydroepiandrosterone or pregnenolone to androstenedione or progesterone, respectively, in placenta, mammary gland, breast tumors, prostate, prostate tumors, and other peripheral tissues. Our earlier studies show that the two enzyme reactions are linked by the coenzyme product, NADH, of the 3 beta-HSD activity. NADH activates the isomerase activity by inducing a time-dependent conformational change in the enzyme protein. The current study tested the hypothesis that the 3 beta-HSD and isomerase activities shared a common coenzyme domain, and it characterized key amino acids that participated in coenzyme binding and the isomerase reaction. Homology modeling with UDP-galactose-4-epimerase predicts that Asp36 is responsible for the NAD(H) specificity of human 3 beta-HSD/isomerase and identifies the Rossmann-fold coenzyme domain at the amino terminus. The D36A/K37R mutant in the potential coenzyme domain and the D241N, D257L, D258L, and D265N mutants in the potential isomerase domain (previously identified by affinity labeling) were created, expressed, and purified. The D36A/K37R mutant shifts the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H), which shows that the two activities utilize a common coenzyme domain. The D257L and D258L mutations eliminate isomerase activity, whereas the D241N and D265N mutants have nearly full isomerase activity. Kinetic analyses and pH dependence studies showed that either Asp257 or Asp258 plays a catalytic role in the isomerization reaction. These observations further characterize the structure/function relationships of human 3 beta-HSD/isomerase 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).


Asunto(s)
Coenzimas/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , NAD/metabolismo , Progesterona Reductasa/química , Progesterona Reductasa/metabolismo , Esteroide Isomerasas/química , Esteroide Isomerasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arginina , Ácido Aspártico , Femenino , Humanos , Cinética , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Mutagénesis Sitio-Dirigida , NAD/química , Placenta/enzimología , Embarazo , Progesterona Reductasa/genética , Conformación Proteica , Proteínas Recombinantes/metabolismo , Esteroide Isomerasas/genética , Especificidad por Sustrato , Células Tumorales Cultivadas
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