Article (Scientific journals)
Human recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties
Lakaye, Bernard; Makarchikov, Alexander F; Wins, Pierre et al.
2004In International Journal of Biochemistry and Cell Biology, 36 (7), p. 1348-1364
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Keywords :
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
Abstract :
[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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Lakaye, Bernard ;  Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique
Makarchikov, Alexander F
Wins, Pierre
Margineanu, Ilca
Roland, Severine
Lins, Laurence  ;  Université de Liège - ULiège > Chimie et bio-industries > Centre de Bio. Fond. - Section de Biologie
Aichour, Ridha
Lebeau, Luc
Elmoualij, Benaïssa ;  Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Histologie humaine
Zorzi, Willy ;  Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Histologie humaine
Coumans, Bernard ;  Université de Liège - ULiège > CNCM/ Centre fac. de rech. en neurobiologie cell. et moléc.
Grisar, Thierry ;  Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique
Bettendorff, Lucien  ;  Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique
More authors (3 more) Less
Language :
English
Title :
Human recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties
Publication date :
2004
Journal title :
International Journal of Biochemistry and Cell Biology
ISSN :
1357-2725
eISSN :
1878-5875
Publisher :
Pergamon-Elsevier Science Ltd, Oxford, United Kingdom
Volume :
36
Issue :
7
Pages :
1348-1364
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 03 November 2010

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