PURIFICATION AND UNUSUAL KINETIC-PROPERTIES OF A TOBACCO ANIONIC PEROXIDASE

The tobacco anionic peroxidase has been isolated from the leaves of tr ansgenic Nicotiana sylvestris plants overproducing this enzyme. The pl ant expression system and the purification protocol developed allow th e preparation of greater than 60 mg of homogeneous enzyme (M(r) 36 kDa , pI 3.5) from 1 kg of fresh leaves, which is an order of magnitude hi gher than for wild-type tobacco plants. The tobacco anionic peroxidase exhibits rather unusual catalytic properties in comparison with horse radish peroxidase (HRP C). Compound I is less active than Compound IZ in the tobacco enzyme. The enzyme is nearly inactive towards iodide, r eflecting the peculiarities of its molecular structure. In particular, the presence of the negatively charged glutamate residue 141 at the e ntrance of the haeme-binding pocket seems to affect the stabilities of Compounds I, II and III, leading to a different enzyme substrate spec ificity than that of HRP C. Investigation of thermal stability towards a number of electron donors reveals the following 'order of stabiliti es': ferrocyanide > guaiacol > 2,2'-azino-bis(3-ethyl-6-benzothiazolin e sulphonate) > iodide > o-dianisidine, which may indicate different b inding sites and rate-limiting steps in the mechanism of the substrate oxidation.