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 Km values of 12.4 μM and 4.7 mM for thiamine and ATP, respectively, and the Vmax value of 360 pmol TDP min−1 mg−1 protein. The enzyme required magnesium ions, and the best phosphate donor was GTP. The purified phosphatase was the dimer of 24 kDa 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 Km values for TDP and TMP were 36 μM and 49 μM, respectively, and the Vmax value for TDP was significantly higher than for TMP (164 versus 60 nmoles min−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.