Enzymes that control the thiamine diphosphate pool in plant tissues. Properties of thiamine pyrophosphokinase and thiamine-(di)phosphate phosphatase purified from Zea mays seedlings.

The pool of thiamine diphosphate (TDP), available for TDP-dependent enzymes involved in the major carbohydrate metabolic pathways, is controlled by two enzyme systems that act in the opposite directions. The thiamine pyrophosphokinase (TPK) activates thiamine into TDP and the numerous phosphatases perform the reverse two-step dephosphorylation of TDP to thiamine monophosphate (TMP) and then to free thiamine. Properties and a possible cooperation of those enzymes in higher plants have not been extensively studied. In this work, we characterize highly purified preparations of TPK and a TDP/TMP phosphatase isolated from 6-day Zea mays seedlings. TPK was the 29-kDa monomeric protein, with the optimal activity at pH 9.0, the K(m) values of 12.4microM and 4.7mM for thiamine and ATP, respectively, and the V(max) value of 360pmol TDPmin(-1)mg(-1) protein. The enzyme required magnesium ions, and the best phosphate donor was GTP. The purified phosphatase was the dimer of 24kDa subunits, showed the optimal activity at pH 5.0 and had a rather broad substrate specificity, although TDP, but not TMP, was one of the preferable substrates. The K(m) values for TDP and TMP were 36microM and 49microM, respectively, and the V(max) value for TDP was significantly higher than for TMP (164 versus 60nmolesmin(-1)mg(-1) protein). The total activities of TPK and TDP phosphatases were similarly decreased when the seedlings were grown under the illumination, suggesting a coordinated regulation of both enzymes to stabilize the pool of the essential coenzyme.

Identification, purification and reconstitution of thiamin metabolizing enzymes in human red blood cells.

Thiamin and its mono- (TMP), di- (TDP) and triphosphate (TTP) were assayed in adult human whole blood using high-performance liquid chromatography (HPLC). TDP and TTP were detected in red blood cells (RBC), but not in plasma. After incubation with 20 microM thiamin and 5 mM glucose for 2 h, the TDP and TTP contents of RBC increased from 111 to 222 and 0.6 to 2.2 nmol/l of packed RBC, respectively, suggesting enzymatic conversion of thiamin to TDP and then to TTP. Thiamin pyrophosphokinase (TPK, EC 2.7.6.2) had not been isolated before from human materials, nor had cytosolic adenylate kinase (AK1, EC 2.7.4.3) in human RBC been demonstrated to catalyze the phosphorylation of TDP to TTP, although AK1 from pig and chicken skeletal muscle possess TTP-synthesizing activity. TPK and AK1 in a human RBC lysate were therefore purified by a series of the conventional techniques. The specific activity of the purified TPK, which was obtained as a single protein, was 720 nmol TDP formed/mg protein per h at 37 degrees C. A partially purified AK1 preparation catalyzed the formation of TTP from TDP (specific activity, 170 nmol/mg protein per h at 37 degrees C) in addition to its proper reaction to form ATP from ADP. After incubation of the purified TPK and AK1 with 20 microM thiamin in the presence of ATP, ADP and Mg2+ at 37 degrees C for 48 h, the amounts of TDP and TTP synthesized were 465 and 54.0 pmol/250 microliters reaction mixture, respectively. Neither TDP nor TTP was formed when TPK was omitted from the reaction mixture and an omission of AK1 resulted in the formation of TDP alone. These results indicate that thiamin is converted to TDP by TPK and, subsequently, to TTP by AK1 in human RBC.

Purification and properties of thiamine pyrophosphokinase in Paracoccus denitrificans.

The existence of thiamine pyrophosphokinase [EC 2.7.6.2] in procaryotic cells was first demonstrated in Paracoccus denitrificans (J. Bacteriol, (1976) 126, 1030-1036). The enzyme was therefore purified from this organism to determine its molecular structure and properties. Thiamine pyrophosphokinase which was purified 620-fold from P. denitrificans showed a single band on both polyacrylamide and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the molecular weight in the latter case was calculated to be 23,000. Gel filtration analysis using Sephadex G-150 gave a molecular weight of 44,000, indicating that this enzyme contains at least two identical subunits. Although sedimentation equilibrium analysis gave a molecular weight of 96,000, indirect evidence suggests that the form having this molecular weight is an aggregate of the functional dimer. The activity of the purified enzyme required thiamine, ATP, and Mg2+, and the enzyme catalyzed thepyrophosphorylation of thiamine by ATP. Km values for thiamine and ATP were 10 microM and 0.38 mM, respectively. The activity was competitively inhibited by pyrithiamine, giving a Ki value of 19 microM. Oxythiamine and chloroethylthiamine were very weak inhibitors of the enzyme. The activity was also inhibited by the product, TPP.

[Isolation and radiometric determination of rat liver ATP: thiamine diphosphate phosphotransferase activity]. / Vydelenie i radiometricheskii metod opredeleniia aktivnosti ATP: tiamindifosfatfosfotransferazy iz pechpeni krys.

A radioassay was developed for estimation of the thiamin diphosphate kinase activity (EC 2.7.4.15); 14C-TDP was used as a substrate. After termination of the reaction (formation of TTP), the products obtained were separated using ion exchange chromatography on SP-Sephadex C-25. The method developed was very sensitive and enabled to estimate the enzymatic activity in tissue homogenates. TDP-kinase was isolated from rat liver with a 70-fold purification. Dependence of the reaction rate on pH value, concentrations of the enzyme and thiamin were studied.