Thiamine triphosphatase

Thiamine triphosphate is present in low concentration in most tissues, but its role remains unknown. Organs and tissues that generate electrical impulses are particularly rich in this compound. 

Thiamine triphosphate, diphosphate, and monophosphate were separated as the thiochrome derivatives on Hamilton PRP-1 column. The mobile phase was 15 mill phosphate buffer (pH 8.5) containing 1% tetrahydrofuran. The solvent flow rate was 0.5 mL/min, and a 20 fiL sample loop was used. Detection was by fluorescence using excitation and emission wavelengths of 365 and 433 nm, respectively. 

The reaction mixture was composed of 50 p.L of membrane preparation, 10 mM Hepes-Tris buffer (pH 6.8), 1.5 mM MgQ2, 1.5 mM EGTA, and 0.1 mM thiamine triphosphate in a total volume of 100 fiL. After 15 minutes of incubation at 25°C, the reaction was stopped by addition of 500 fiL of 6% trichloroacetic acid. The supemate obtained by centrifugation was extracted with 4 volumes of diethyl ether. The thiamine derivatives were transformed into fluorescent thiochromes by the addition of 50 fiL of oxidant [4.3 mJW K3Fe(CN)6 in 15% NaOH] to 80 fiL of sample. Thiamine diphosphatase activity was minimized by using magnesium as the metal and EGTA to chelate calcium. 

The source of enzyme was crude or partially purified membranes from the main electrical organ of Electrophorus electricus. 

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In both muscle and the central nervous system, there is an active thiamin triphosphatase, so that tissue concentrations of thiamin triphosphate are strictly regulated (Nishino et al., 1983 Miyoshi et al., 1990 Lakaye et al., 2002). 

Thiamin triphosphate is formed in brain and skeletal muscle by phosphorylation of thiamin diphosphate (Section 6.2), and its concentration is very precisely controlled, because there is also an active thiamin triphosphatase (Lakaye et al., 2002). In nervous tissue thiamin triphosphate is localized... 

Lakaye B, Makarchikov AF, Fernandes Antunes A, Zorzi W, Coumans B, De Pauw E, Wins P, Grisar T, and Bettendorff L (2002) Molecular characterization of a specific thiamine triphosphatase widely expressed in mammalian tissues./oMmfl/o/Biotogtcfl/ Chemistry 277,13771-7. 

The three tissue enzymes known to participate in formation of the phosphate esters are (1) thiaminokinase (a pyro-phosphokinase), which catalyzes formation of TPP and adenosine monophosphate (AMP) from thiamine and adenosine triphosphate (ATP) (2) TPP-ATP phosphoryl-transferase (cytosoHc 5"-adenylic kinase)which forms the triphosphate and adenosine diphosphate from TPP and ATP and (3) thiamine triphosphatase, which hydrolyzes TPP to the monophosphate. Although thiaminokinase is widespread, the phosphoryl transferase and membrane-associated triphosphatase are mainly in nervous tissue. 

Thiamine derivatives inhibit glucose synthesis, transketolase (yeast), phosphodiesterase (snake venom), thiamine triphosphatase, thymidylate kinase. 

Thiamin phosphate derivatives can be readily interconverted (Figure 5.5), but the enzymes involved in these reactions remain poorly charaeterized. Only two of them, the essential thiamin pyrophosphokinase and the 25 kDa thiamin triphosphatase, have been charaeterized at the molecular level. 

The catalytic domains of human 25 kDa hThTPase and CyaB-like adenylyl cyclase from Aeromonas hydrophilia define a novel superfamily of domains that should bind organic phosphates (Iyer and Aravind 2002). This superfamily of proteins was therefore called CYTH (CYaB-THiamin triphosphatase), and the presence of orthologs was demonstrated in all three superkingdoms of life. This suggested that CYTH is an ancient enzymatic domain and that a representative must have been present in the last universal common ancestor (LUCA) of all extant life forms. It appears that the CYTH superfamily includes enzymes with various catalytic properties (adenylyl cyclase or inorganic triphosphatase in some bacteria, RNA triphosphatase in yeast, ThTPase in animals) but with important common features ... 

ThTP is synthesized by a chemiosmotic mechanism from ThDP and inorganic phosphate and is hydrolysed by a specific soluble thiamin triphosphatase. 

Iyer, L.M., and Aravind, L., 2002. The catalytic domains of thiamine triphosphatase and CyaB-like adenylyl cyclase define a novel superfamily of domains that bind organic phosphates. BMC Genomics. 3 33. 

Makarchikov, A.F., Lakaye, B., Gulyai, I.E., Czerniecki, J., Coumans, B., Wins, P., Grisar, T., and Bettendorff, L., 2003. Thiamine triphosphate and thiamine triphosphatase activities from bacteria to mammals. Cellular and Molecular Life Sciences. 60 1477-1488. 

Song, J., Bettendorff, L., Tonelli, M., and Markley, J.L., 2008. Structural basis for the catalytic mechanism of mammalian 25-kDa thiamine triphosphatase. Journal of Biological Chemistry. 283 10939-10948. 

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