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.
Rapala-Kozik
Maria
M
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland. rapala@mol.uj.edu.pl
Gołda
Anna
A
Kujda
Marta
M
D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D036226:Seedlings / Q000201:enzymology*; D013836:Thiamin Pyrophosphokinase / Q000302:isolation & purification* / Q000378:metabolism*; D013835:Thiamine Pyrophosphate / Q000378:metabolism*; D003313:Zea mays / Q000201:enzymology*
D013836:Thiamin Pyrophosphokinase; D010744:Phosphoric Monoester Hydrolases; D013835:Thiamine Pyrophosphate