Pig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: insight in the catalytic mechanisms of this enzyme.
Thiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian 25-kDa thiamine triphosphatase. The enzyme was active in all mammalian species studied except pig, although the corresponding mRNA was present. In order to determine whether the very low ThTPase activity in pig tissues is due to the absence of the protein or to a lack of catalytic efficiency, we expressed human and pig ThTPase in E. coli as GST fusion proteins. The purified recombinant pig GST-ThTPase was found to be 2-3 orders of magnitude less active than human GST-ThTPase. Using site-directed mutagenesis, we show that, in particular, the change of Glu85 to lysine is responsible for decreased solubility and catalytic activity of the pig enzyme. Immunohistochemical studies revealed a distribution of the protein in pig brain very similar to the one reported in rodent brain. Thus, our results suggest that a 25-kDa protein homologous to hThTPase but practically devoid of enzyme activity is expressed in pig tissues. This raises the possibility that this protein may play a physiological role other than ThTP hydrolysis.
Center for Cellular and Molecular Neurobiology, University of Liège, B-4000 Liège, Belgium.
Piotr
P
Szyniarowski
Bernard
B
Lakaye
Jan
J
Czerniecki
Alexander F
AF
Makarchikov
Pierre
P
Wins
Ilca
I
Margineanu
Bernard
B
Coumans
Thierry
T
Grisar
Lucien
L
Bettendorff
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013552:Swine; D013837:Thiamin-Triphosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D013837:Thiamin-Triphosphatase