دورية أكاديمية
Engineering the properties of a cold active enzyme through rational redesign of the active site.
| العنوان: | Engineering the properties of a cold active enzyme through rational redesign of the active site. |
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| المؤلفون: | Tsigos I; Institute of Molecular Biology and Biotechnology (IMBB), Enzyme Technology Division, Heraklion 711 10, Crete, Greece., Mavromatis K, Tzanodaskalaki M, Pozidis C, Kokkinidis M, Bouriotis V |
| المصدر: | European journal of biochemistry [Eur J Biochem] 2001 Oct; Vol. 268 (19), pp. 5074-80. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies Country of Publication: England NLM ID: 0107600 Publication Model: Print Cited Medium: Print ISSN: 0014-2956 (Print) Linking ISSN: 00142956 NLM ISO Abbreviation: Eur J Biochem Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: -2004: Oxford, UK : Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies Original Publication: Berlin, New York, Springer. |
| مواضيع طبية MeSH: | Alkaline Phosphatase/*metabolism, Alkaline Phosphatase/antagonists & inhibitors ; Alkaline Phosphatase/chemistry ; Alkaline Phosphatase/genetics ; Base Sequence ; Binding Sites ; DNA Primers ; Kinetics ; Models, Molecular ; Mutagenesis, Site-Directed ; Temperature |
| مستخلص: | In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side-chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild-type in the temperature range 5-25 degrees C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5-15 degrees C and a significantly increased activity at 20-25 degrees C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135-->Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild-type sequence, which may affect active site flexibility through changes in various conformational constraints. |
| المشرفين على المادة: | 0 (DNA Primers) EC 3.1.3.1 (Alkaline Phosphatase) |
| تواريخ الأحداث: | Date Created: 20011009 Date Completed: 20011204 Latest Revision: 20190620 |
| رمز التحديث: | 20221213 |
| DOI: | 10.1046/j.0014-2956.2001.02432.x |
| PMID: | 11589698 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 0014-2956 |
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| DOI: | 10.1046/j.0014-2956.2001.02432.x |