Reference : Human recombinant thiamine triphosphatase: purification, secondary structure and catalyt...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/4630
Human recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties
English
Lakaye, Bernard mailto [Université de Liège - ULg > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique >]
Makarchikov, Alexander F [> > > >]
Wins, Pierre [> > > >]
Margineanu, Ilca [> > > >]
Roland, Severine [> > > >]
Lins, Laurence mailto [Université de Liège - ULg > > >Chimie et bio-industries > Centre de Bio. Fond. - Section de Biologie > >]
Aichour, Ridha [> > > >]
Lebeau, Luc [> > > >]
Elmoualij, Benaïssa mailto [Université de Liège - ULg > Département des sciences biomédicales et précliniques > Histologie humaine >]
Zorzi, Willy mailto [Université de Liège - ULg > Département des sciences biomédicales et précliniques > Histologie humaine >]
Coumans, Bernard mailto [Université de Liège - ULg > > CNCM/ Centre fac. de rech. en neurobiologie cell. et moléc. >]
Grisar, Thierry mailto [Université de Liège - ULg > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique]
Bettendorff, Lucien mailto [Université de Liège - ULg > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique >]
2004
International Journal of Biochemistry & Cell Biology
Pergamon-Elsevier Science Ltd
36
7
1348-1364
Yes (verified by ORBi)
International
1357-2725
Oxford
United Kingdom
[en] thiamine triphosphate ; Fourier-transform infrared spectroscopy ; zinc ions ; diethylpyrocarbonate ; Woodward's reagent K
Cloning, Molecular ; DNA, Complementary/genetics ; Diethyl Pyrocarbonate/chemistry ; Enzyme Activation ; Enzyme Stability ; Escherichia coli/enzymology/genetics ; Molecular Structure ; Substrate Specificity
[en] 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.
Researchers ; Professionals
http://hdl.handle.net/2268/4630
also: http://hdl.handle.net/2268/65878
10.1016/j.biocel.2003.11.013

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