Mutations in putative glycosylation sites of rat 11β-hydroxysteroid dehydrogenase affect enzymatic activity

التفاصيل البيبلوغرافية
العنوان: Mutations in putative glycosylation sites of rat 11β-hydroxysteroid dehydrogenase affect enzymatic activity
المؤلفون: Anil K. Agarwal, Perrin C. White, Carl Monder, Tomoatsu Mune
المصدر: Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1248:70-74
بيانات النشر: Elsevier BV, 1995.
سنة النشر: 1995
مصطلحات موضوعية: Glycosylation, Molecular Sequence Data, Biophysics, Dehydrogenase, CHO Cells, Reductase, Biology, Transfection, Biochemistry, Isozyme, Amidohydrolases, Serine, chemistry.chemical_compound, Structural Biology, Cricetinae, Animals, Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase, Amino Acid Sequence, Asparagine, Molecular Biology, Short-chain dehydrogenase, Hydroxysteroid Dehydrogenases, Molecular biology, Rats, chemistry, Mutagenesis, Site-Directed, 11-beta-Hydroxysteroid Dehydrogenases, Branched-chain alpha-keto acid dehydrogenase complex, Sequence Alignment
الوصف: 11 β-hydroxysteroid dehydrogenase (11-HSD) catalyzes the interconversion of corticosterone and 11-dehydrocorticosterone in rats, or cortisol and cortisone in humans. The ‘liver’ or ‘Type I’ isozyme is a widely distributed glycoprotein that utilizes NADP + as a co-factor. To study the role of glycosylation in maintaining enzymatic activity, we introduced mutations into the two potential N -linked glycosylation sites (asparagine- X -serine, residues 158–160 and 203–205) predicted from the rat cDNA sequence. Mutagenesis was performed by a PCR based technique, and wild-type (WT) and mutant cDNAs were expressed in Chinese hamster ovary cells after cloning into the pCMV4 vector. At each putative glycosylation site, asparagine (N) was changed to glutamine (Q) or aspartic acid (D), and serine (S) changed to alanine (A). All three modifications of the first site (N158Q, N158D, S160A) had minimal (75–100% of WT) effects on dehydrogenase activity and caused a mild (50–75% of WT) decrease in reductase activity. In contrast, mutations at the second site had marked effects, with N203Q and N203D completely abolishing both dehydrogenase and reductase activities and S205A decreasing both activities to about 20% of WT. The double mutation of S160A and S205A also abolished all activity, even though the enzyme carrying each mutation alone was, at least, partially active. The results suggest that N203 (which is highly but not completely conserved in short chain dehydrogenase enzymes) is essential for activity of 11-HSD. N -linked glycosylation may be necessary for full activity or stability of the enzyme.
تدمد: 0167-4838
URL الوصول: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::5e924c5d6c25beeafe2e76f1933d57ea
https://doi.org/10.1016/0167-4838(95)00005-f
حقوق: CLOSED
رقم الأكسشن: edsair.doi.dedup.....5e924c5d6c25beeafe2e76f1933d57ea
قاعدة البيانات: OpenAIRE