[The rate of molecular evolution of mammalian soluble thiamine triphosphatase].

Thiamine triphosphate (ThTP) exists in various living cells--from bacteria to mammals. ThTP concentration in mammals is regulated by a specific soluble ThTPase, which has not been revealed experimentally, however, in other organisms. In NCBI and Ensembl databases we have found information about full-size or partial amino acid sequences of the enzyme from 38 mammal species. An average rate of amino acid substitutions (k(aa)) in ThTPase molecule was estimated from the data available to be 1.41 x 10(-9) per site per year. This corresponds to unit evolutionary period of about 4.4 million years. The evolutionary rate varies for different portions of the enzyme, C-terminal fragment being the most variable (k(aa) = 3.76 x 10(-9); calculated only for 230 aa species + elephant). An average replacement rate of 1.95 x 10(-9) per amino acid site per year was calculated for the central portion of the enzyme (residues 69-141), while N-terminal sequence (residues 1-68) and 142-210 fragment evolved with k(aa) of 1.03 x 10(-9) and 0.81 x 10(-9), respectively.

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.

Expression of 25 kDa thiamine triphosphatase in rodent tissues using quantitative PCR and characterization of its mRNA.

Thiamine triphosphate (ThTP) is found in most organisms, but its biological role remains unclear. In mammalian tissues, cellular ThTP concentrations remain low, probably because of hydrolysis by a specific 25 kDa thiamine triphosphatase (ThTPase). The aim of the present study was to use quantitative PCR, for comparing the 25 kDa ThTPase mRNA expression in various mouse tissues with its enzyme activities. ThTPase mRNA was expressed at only a few copies per cell. The highest amount of mRNA was found in testis, followed by lung and muscle, while the highest enzyme activities were found in liver and kidney. The poor correlation between mRNA levels and enzyme activities might result either from tissue-specific post-transcriptional regulation of mRNA processing and/or translation or from the regulation of enzyme activities by post-translational mechanisms. Purified recombinant human ThTPase was phosphorylated by casein kinase II, but this phosphorylation did not modify the enzyme activity. However, the characterization of the 3'-untranslated mRNA region revealed a unique, highly conserved, 200-nucleotide sequence that might be involved in translational control. In situ hybridization studies in testis suggest a predominant localization of ThTPase mRNA in poorly differentiated spermatogenic cells. This is the first study demonstrating a cell-specific 25 kDa ThTPase mRNA expression, suggesting that this enzyme might be related to the degree of differentiation or the metabolic state of the cell.

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.