Purification and Properties of Thiamine Pyrophosphokinase in Paracoccus denitrificans

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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 the pyrophosphorylation of thiamine by ATP. K_m values for thiamine and ATP were 10 μM and 0.38mM, respectively. The activity was competitively inhibited by pyrithiamine, giving a K₁ value of 19 μM. Oxythiamine and chloroethylthiamine were very weak inhibitors of the enzyme. The activity was also inhibited by the product, TPP.