کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8339793 | 1541044 | 2010 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
The functions of key residues in the inhibitor, substrate and cofactor sites of human 3β-hydroxysteroid dehydrogenase type 1 are validated by mutagenesis
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: The functions of key residues in the inhibitor, substrate and cofactor sites of human 3β-hydroxysteroid dehydrogenase type 1 are validated by mutagenesis The functions of key residues in the inhibitor, substrate and cofactor sites of human 3β-hydroxysteroid dehydrogenase type 1 are validated by mutagenesis](/preview/png/8339793.png)
چکیده انگلیسی
In postmenopausal women, human 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors, while 3β-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to determine if Arg195 in 3β-HSD1 vs. Pro195 in 3β-HSD2 in the substrate/inhibitor binding site is a critical structural difference responsible for the higher affinity of 3β-HSD1 for inhibitor and substrate steroids compared to 3β-HSD2 and whether Asp61, Glu192 and Thr8 are fingerprint residues for cofactor and substrate binding using site-directed mutagenesis. The R195P-1 mutant of 3β-HSD1 and the P195R-2 mutant of 3β-HSD2 have been created, expressed, purified and characterized kinetically. Dixon analyses of the inhibition of the R195P-1 mutant, P195R-2 mutant, wild-type 3β-HSD1 and wild-type 3β-HSD2 by trilostane has produced kinetic profiles that show inhibition of 3β-HSD1 by trilostane (Ki = 0.10 μM, competitive) with a 16-fold lower Ki and different mode than measured for 3β-HSD2 (Ki = 1.60 μM, noncompetitive). The R195P-1 mutation shifts the high-affinity, competitive inhibition profile of 3β-HSD1 to a low-affinity (trilostane Ki = 2.56 μM), noncompetitive inhibition profile similar to that of 3β-HSD2 containing Pro195. The P195R-2 mutation shifts the low-affinity, noncompetitive inhibition profile of 3β-HSD2 to a high-affinity (trilostane Ki = 0.19 μM), competitive inhibition profile similar to that of 3β-HSD1 containing Arg195. Michaelis-Menten kinetics for DHEA, 16β-hydroxy-DHEA and 16α-hydroxy-DHEA substrate utilization by the R195P-1 and P195R-2 enzymes provide further validation for higher affinity binding due to Arg195 in 3β-HSD1. Comparisons of the Michaelis-Menten values of cofactor and substrate for the targeted mutants of 3β-HSD1 (D61N, D61V, E192A, T8A) clarify the functions of these residues as well.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: The Journal of Steroid Biochemistry and Molecular Biology - Volume 120, Issues 4â5, June 2010, Pages 192-199
Journal: The Journal of Steroid Biochemistry and Molecular Biology - Volume 120, Issues 4â5, June 2010, Pages 192-199
نویسندگان
James L. Thomas, Vance L. Mack, Jingping Sun, J. Ross Terrell, Kevin M. Bucholtz,