Human recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties
| Autor: | Bernard Lakaye, Benaissa El Moualij, Ridha Aichour, Laurence Lins, Ilca Margineanu, Pierre Wins, Willy Zorzi, Thierry Grisar, Bernard Coumans, Lucien Bettendorff, Luc Lebeau, Alexander F Makarchikov, Séverine Roland |
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| Přispěvatelé: | Center for Cellular and Molecular Neurobiology, Universi de Lge, Chimie et Biologie des Membranes et des Nanoobjets (CBMN), Université de Bordeaux (UB)-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Conception et application de molécules bioactives (CAMB), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Institute of Human Histology-CRPP (CRPP), Université de Liège |
| Rok vydání: | 2004 |
| Předmět: |
DNA
Complementary Cations Divalent [SDV]Life Sciences [q-bio] Thiamin-Triphosphatase Regulatory site Thiamine Triphosphate Biochemistry Catalysis Protein Structure Secondary Substrate Specificity 03 medical and health sciences Enzyme activator chemistry.chemical_compound Adenosine Triphosphate Thiamine triphosphatase Cerebellum Diethyl Pyrocarbonate Enzyme Stability Escherichia coli Humans Cloning Molecular Binding site ComputingMilieux_MISCELLANEOUS 030304 developmental biology chemistry.chemical_classification 0303 health sciences Binding Sites Molecular Structure biology Chemistry 030302 biochemistry & molecular biology Active site Cell Biology Recombinant Proteins Enzyme Activation Enzyme biology.protein Thiamine triphosphate |
| Zdroj: | The International Journal of Biochemistry & Cell Biology The International Journal of Biochemistry & Cell Biology, 2004, 36 (7), pp.1348-1364. ⟨10.1016/j.biocel.2003.11.013⟩ |
| ISSN: | 1357-2725 |
| DOI: | 10.1016/j.biocel.2003.11.013 |
| Popis: | Thiamine triphosphate (ThTP) is found in most living organisms and it may act as a phosphate donor for protein phosphorylation. We have recently cloned the cDNA coding for a highly specific mammalian 25 kDa thiamine triphosphatase (ThTPase; EC 3.6.1.28). As the enzyme has a high catalytic efficiency and no sequence homology with known phosphohydrolases, it was worth investigating its structure and catalytic properties. For this purpose, we expressed the untagged recombinant human ThTPase (hThTPase) in E. coli, produced the protein on a large scale and purified it to homogeneity. Its kinetic properties were similar to those of the genuine human enzyme, indicating that the recombinant hThTPase is completely functional. Mg2+ ions were required for activity and Ca2+ inhibited the enzyme by competition with Mg2+. With ATP as substrate, the catalytic efficiency was 10(-4)-fold lower than with ThTP, confirming the nearly absolute specificity of the 25 kDa ThTPase for ThTP. The activity was maximum at pH 8.5 and very low at pH 6.0. Zn2+ ions were inhibitory at micromolar concentrations at pH 8.0 but activated at pH 6.0. Kinetic analysis suggests an activator site for Mg2+ and a separate regulatory site for Zn2+. The effects of group-specific reagents such as Woodward's reagent K and diethylpyrocarbonate suggest that at least one carboxyl group in the active site is essential for catalysis, while a positively charged amino group may be involved in substrate binding. The secondary structure of the enzyme, as determined by Fourier-transform infrared spectroscopy, was predominantly beta-sheet and alpha-helix. |
| Databáze: | OpenAIRE |
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