Purification and properties of threonine dehydratase from Rhodopseudomonas spheroides

Biosynthetic threonine dehydratase (l-threonine hydro-lyase (deaminating), EC 4.2.1.16) has been purified about 3000-fold from cell-free extracts of the photosynthetic bacterium, Rhodopseudomonas spheroides. The addition of dithiothreitol, pyridoxal phosphate and allothreonine was essential for stabilization of the enzyme during both purification and storage. The molecular weight of the enzyme was estimated to be in the vicinity of 200 000. Kinetic studies indicated that, at low concentrations of threonine, there is a time-dependent loss of enzymic activity which is influenced by the pH and composition of buffers, as well as by KCl and pyridoxal phosphate. In the presence of isoleucine, there is a relatively slow development of the steady-state inhibited rate. No lag period was associated with the inhibition of the reaction by higher concentrations of allothreonine or with the activation by lower concentrations of this compound. It has been concluded that the time-dependent inhibition by isoleucine can be accounted for on the assumption that this reactant induces the enzyme to undergo isomerization reactions which are slow relative to the rate of catalysis.