title: Human recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties.
abstract: 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.
authors.affiliation: Center for Cellular and Molecular Neurobiology, University of Liège, 17 place Delcour, B-4020 Liège, Belgium.
authors.forename: Bernard
authors.initials: B
authors.lastname: Lakaye
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Makarchikov
authors.forename: Pierre
authors.initials: P
authors.lastname: Wins
authors.forename: Ilca
authors.initials: I
authors.lastname: Margineanu
authors.forename: Séverine
authors.initials: S
authors.lastname: Roland
authors.forename: Laurence
authors.initials: L
authors.lastname: Lins
authors.forename: Ridha
authors.initials: R
authors.lastname: Aichour
authors.forename: Luc
authors.initials: L
authors.lastname: Lebeau
authors.forename: Benaïssa
authors.initials: B
authors.lastname: El Moualij
authors.forename: Willy
authors.initials: W
authors.lastname: Zorzi
authors.forename: Bernard
authors.initials: B
authors.lastname: Coumans
authors.forename: Thierry
authors.initials: T
authors.lastname: Grisar
authors.forename: Lucien
authors.initials: L
authors.lastname: Bettendorff
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry; D002531:Cerebellum / Q000201:enzymology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D015394:Molecular Structure; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity; D013837:Thiamin-Triphosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013838:Thiamine Triphosphate / Q000031:analogs & derivatives
chemical_list: D002413:Cations, Divalent; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013838:Thiamine Triphosphate; D000255:Adenosine Triphosphate; D013837:Thiamin-Triphosphatase; D004047:Diethyl Pyrocarbonate