Human recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties.
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.
Lakaye
Bernard
B
Center for Cellular and Molecular Neurobiology, University of Liège, 17 place Delcour, B-4020 Liège, Belgium.
Makarchikov
Alexander F
AF
Wins
Pierre
P
Margineanu
Ilca
I
Roland
Séverine
S
Lins
Laurence
L
Aichour
Ridha
R
Lebeau
Luc
L
El Moualij
Benaïssa
B
Zorzi
Willy
W
Coumans
Bernard
B
Grisar
Thierry
T
Bettendorff
Lucien
L
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
D002413:Cations, Divalent; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013838:Thiamine Triphosphate; D000255:Adenosine Triphosphate; D013837:Thiamin-Triphosphatase; D004047:Diethyl Pyrocarbonate