Gazaryan I G, Lagrimini L M
Department of Horticulture and Crop Sciences, Ohio State University, Columbus 43210-1096, USA.
Phytochemistry. 1996 Mar;41(4):1029-34. doi: 10.1016/0031-9422(95)00779-2.
The tobacco anionic peroxidase has been isolated from the leaves of transgenic Nicotiana sylvestris plants overproducing this enzyme. The plant expression system and the purification protocol developed allow the 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 higher than for wild-type tobacco plants. The tobacco anionic peroxidase exhibits rather unusual catalytic properties in comparison with horseradish peroxidase (HRP C). Compound I is less active than Compound II in the tobacco enzyme. The enzyme is nearly inactive towards iodide, reflecting the peculiarities of its molecular structure. In particular, the presence of the negatively charged glutamate residue 141 at the entrance of the haeme-binding pocket seems to affect the stabilities of Compounds I, II and III, leading to a different enzyme substrate specificity than that of HRP C. Investigation of thermal stability towards a number of electron donors reveals the following 'order of stabilities': ferrocyanide > guaiacol > 2,2'-azino-bis(3-ethyl-6- benzothiazoline sulphonate) > iodide > o-dianisidine, which may indicate different binding sites and rate-limiting steps in the mechanism of the substrate oxidation.
烟草阴离子过氧化物酶是从过量表达该酶的转基因野生烟草植株的叶片中分离得到的。所开发的植物表达系统和纯化方案能够从1千克鲜叶中制备出超过60毫克的纯酶（分子量36 kDa，等电点3.5），这比野生型烟草植株的产量高出一个数量级。与辣根过氧化物酶（HRP C）相比，烟草阴离子过氧化物酶表现出相当不寻常的催化特性。在烟草酶中，化合物I的活性低于化合物II。该酶对碘化物几乎无活性，这反映了其分子结构的特殊性。特别是，血红素结合口袋入口处带负电荷的谷氨酸残基141的存在似乎影响了化合物I、II和III的稳定性，导致其酶底物特异性与HRP C不同。对多种电子供体的热稳定性研究揭示了以下“稳定性顺序”：亚铁氰化物>愈创木酚>2,2'-联氮-双-(3-乙基苯并噻唑啉-6-磺酸)>碘化物>邻联茴香胺，这可能表明底物氧化机制中存在不同的结合位点和限速步骤。
