کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1939307 1050758 2007 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Mutagenesis of non-conserved active site residues improves the activity and narrows the specificity of human thymidine kinase 2
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Mutagenesis of non-conserved active site residues improves the activity and narrows the specificity of human thymidine kinase 2
چکیده انگلیسی

Human thymidine kinase 2 (TK2) is critical for the nucleotide salvage pathway and phosphorylation of nucleoside analog prodrugs in vivo; however, it remains poorly studied because of difficulties in expressing it heterologously. TK2 is strictly pyrimidine-specific, whereas its phylogenetic relative, the Drosophila melanogaster deoxyribonucleoside kinase (DmdNK), shows higher activity and broader specificity towards both pyrimidines and purines. These differences are counterintuitive, as only two of 29 active site residues differ in the two enzymes: F80 and M118 in DmdNK are L78 and L116 in TK2. In addition to reporting an optimized protocol for the expression and purification of TK2, we have used site-directed mutagenesis to introduce the DmdNK-like amino acids into TK2, and characterized the three resulting enzymes (L78F-TK2, L116M-TK2, and L78F/L116M-TK2). These mutations improve the KM for thymidine, increasing the catalytic activity of L78F/L116M-TK2 4.4-fold, yet leaving the activity for deoxycytidine or the purine nucleosides unchanged.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochemical and Biophysical Research Communications - Volume 354, Issue 3, 16 March 2007, Pages 802–807
نویسندگان
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