Pig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: Insight in the catalytic mechanisms of this enzyme
Institute of Biology, University of Bialystok, Belostok, Podlasie, PolandBiochimica et Biophysica Acta (Impact Factor: 4.66). 09/2005; 1725(1):93-102. DOI: 10.1016/j.bbagen.2005.05.026
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.
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- "This could be the reason why no soluble ThTPase activity was observed in fish tissues (Makarchikov et al. 2003). Surprisingly, and due a single amino acid substitution , the pig has a low catalytic activity, though it seems to be expressed according to a similar pattern than in rodent tissues (Szyniarowski et al. 2005). This means that in pig, bird and fish tissues, 25-kDa ThTPase is either absent or its catalytic activity is very low, allowing ThTP to accumulate (Table 1). "
ABSTRACT: Thiamine triphosphate (ThTP) was discovered over 60 years ago and it was long thought to be a specifically neuroactive compound. Its presence in most cell types, from bacteria to mammals, would suggest a more general role but this remains undefined. In contrast to thiamine diphosphate (ThDP), ThTP is not a coenzyme. In E. coli cells, ThTP is transiently produced in response to amino acid starvation, while in mammalian cells, it is constitutively produced at a low rate. Though it was long thought that ThTP was synthesized by a ThDP:ATP phosphotransferase, more recent studies indicate that it can be synthesized by two different enzymes: (1) adenylate kinase 1 in the cytosol and (2) FoF1-ATP synthase in brain mitochondria. Both mechanisms are conserved from bacteria to mammals. Thus ThTP synthesis does not seem to require a specific enzyme. In contrast, its hydrolysis is catalyzed, at least in mammalian tissues, by a very specific cytosolic thiamine triphosphatase (ThTPase), controlling the steady-state cellular concentration of ThTP. In some tissues where adenylate kinase activity is high and ThTPase is absent, ThTP accumulates, reaching ≥ 70 % of total thiamine, with no obvious physiological consequences. In some animal tissues, ThTP was able to phosphorylate proteins, and activate a high-conductance anion channel in vitro. These observations raise the possibility that ThTP is part of a still uncharacterized cellular signaling pathway. On the other hand, its synthesis by a chemiosmotic mechanism in mitochondria and respiring bacteria might suggest a role in cellular energetics.
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- "intense elec - trical activity . Thus , TTP seems to be essentially associated with neurons : In rat brain , the amount of TTP is about five times higher in neurons than in astrocytes ( Bettendorff et al . 1991 ) . Indeed , the membrane - associated enzyme form ( TTPase ) may play a physiologic role other than TTP hydrolysis in mammalian tissues ( Szyniarowski et al . 2005 ) . In vertebrate tissues , TTPase may act as a phosphate donor for the phosphorylation of certain proteins ; that may be part of a new signal transduction pathway ( Czerniecki et al . 2004 ) . Thiamine deficiency decreases membrane - associated TTPase activity ( Iwata et al . 1974 ) . Membrane - associated TTPase is affected by proteol"
ABSTRACT: In the literature, previous descriptions of the role of thiamine (B1 vitamin) focused mostly on its biochemical functions as a coenzyme precursor of some key enzymes of the carbohydrate metabolism. This report reviews recent developments on the metabolic and structural role of thiamine, e.g., the coenzyme and noncoenzyme functions of the vitamin. Taking into account analysis of our experimental data relating to the effects of thiamine deficiency on developing central nervous system (CNS) and data available in literature, we seek to establish a clear difference between the metabolic and structural role of thiamine. Our experimental data indicate that the specific and nonspecific effects express two diametrically diverse functions of thiamine in development: the nonspecific effects show up the metabolic consequences of thiamine deficiency resulting in apoptosis and severe cellular deficit; inversely, the specific effects announced the structural consequences of thiamine deficiency, described as cellular membrane damage, irregular and ectopic cells. The review highlights the existence of noncoenzyme functions of this vitamin through its interactions with biological membranes.
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- "Also, it is probable that in skeletal muscle where AK1 activity is very high, the latter may contribute to a significant synthesis of cytosolic ThTP, especially in those species where soluble ThTPase activity is absent such as in electric organ [25,26], chicken  and pig skeletal muscle . Indeed, electric organs and bird tissues contain no soluble ThTPase and pig tissues express a catalytically inefficient ThTPase . "
ABSTRACT: Thiamine triphosphate (ThTP) exists in most organisms and might play a role in cellular stress responses. In E. coli, ThTP is accumulated in response to amino acid starvation but the mechanism of its synthesis is still a matter of controversy. It has been suggested that ThTP is synthesized by an ATP-dependent specific thiamine diphosphate kinase. However, it is also known that vertebrate adenylate kinase 1 catalyzes ThTP synthesis at a very low rate and it has been postulated that this enzyme is responsible for ThTP synthesis in vivo. Here we show that bacterial, as vertebrate adenylate kinases are able to catalyze ThTP synthesis, but at a rate more than 106-fold lower than ATP synthesis. This activity is too low to explain the high rate of ThTP accumulation observed in E. coli during amino acid starvation. Moreover, bacteria from the heat-sensitive CV2 strain accumulate high amounts of ThTP (>50% of total thiamine) at 37 degrees C despite complete inactivation of adenylate kinase and a subsequent drop in cellular ATP. These results clearly demonstrate that adenylate kinase is not responsible for ThTP synthesis in vivo. Furthermore, they show that E. coli accumulate large amounts of ThTP under severe energy stress when ATP levels are very low, an observation not in favor of an ATP-dependent mechanisms for ThTP synthesis.
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