5‐Oxo‐prolinase in Nicotiana tabacum: catalytic properties and subcellular localization

5‐Oxo‐prolinase of cultured tobacco cells is a soluble enzyme predominantly localized in the cytoplasm. To get optimal enzyme activity, the presence of the monovalent cation ammonium and the divalent cations Mg2+ and Mn2+ in the assay mixture is necessary. The enzyme has an extremely alkaline pH—(9.5–10.5) and a high temperature ‐ optimum (55°C). In contrary to the 5‐oxo‐prolinase from animal cells, where heat‐stabilization by 5‐oxo‐proline is observed, the high temperature optimum of the tobacco enzyme is due to stabilization by ATP. High 5‐oxo‐prolinase activity in tobacco cell homogenates was not only shown with the co‐substrate ATP, but with other purine‐nucleotides, too, although ATP was the best co‐substrate of the compounds tested. Substrate affinity of the tobacco enzyme (Km 5‐oxo‐proline = 30.5 μM) is similar to that demonstrated for wheat germ 5‐oxo‐prolinase. Competitive inhibition by the 5‐oxo‐proline analogues 2‐imidazolidone‐4‐carboxylic acid(K1= 14.5 μM) and dihydroorotic acid (K1=2 mM) revealed a much higher sensitivity of tobacco 5‐oxo‐prolinase to these compounds than observed for the mammalian enzyme.