Pigment-free NADPH:protochlorophyllide oxidoreductase from Avena sativa L -: Purification and substrate specificity
Pigment-free NADPH:protochlorophyllide oxidoreductase from Avena sativa L -: Purification and substrate specificity
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DOI：
10.1046/j.1432-1327.1999.00627.x
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发表时间：
1999-11-01
期刊：
影响因子：
--
通讯作者：
Rüdiger, W
中科院分区：
文献类型：
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作者：
Klement, H;Helfrich, M;Rüdiger, W
NADPH：原叶绿素氧化还原酶(POR)是光依赖的叶绿素生物合成的关键酶。它在黑暗生长的植物中以POR-原叶绿素a-NADPH三元酶-底物复合体的形式积累。在这里，我们描述了一种从燕麦幼苗的黄体中纯化无色素FOR的简单方法。该方法包括N-辛基-β-D-葡萄糖苷的差示增溶和DEAE-纤维素的一个层析步骤。我们用色素和蛋白质分析表明，黄体含有For和原叶绿素a的一对一的复合体。这些类似物在大环的侧链上有所不同，部分含有锌而不是中心镁。A环或B环具有不同侧链的6个类似物是活性底物，D或E环具有不同侧链的7个类似物不被FOR接受为底物。光依赖反应的动力学表现为三组底物类似物，分别为快、中、慢反应。为了评估动力学数据，必须测定反应缓冲液中的摩尔消光系数。当浓度大于2摩尔底物/摩尔酶时，对原叶绿素a及其类似物有抑制作用。
The enzyme NADPH:protochlorophyllide oxidoreductase (POR) is the key enzyme for light-dependent chlorophyll biosynthesis. It accumulates in dark-grown plants as the ternary enzyme-substrate complex POR-protochlorophyllide a-NADPH. Here, we describe a simple procedure for purification of pigment-free FOR from etioplasts of Avena sativa seedlings. The procedure implies differential solubilization with n-octyl-beta-D-glucoside and one chromatographic step with DEAE-cellulose. We show, using pigment and protein analysis, that etioplasts contain a one-to-one complex of FOR and protochlorophyllide a. The preparation of 13 analogues of protochlorophyllide a is described. The analogues differ in the side chains of the macrocycle and in part contain zinc instead of the central magnesium. Six analogues with different side chains at rings A or B are active substrates, seven analogues with different side chains at rings D or E are not accepted as substrates by FOR. The kinetics of the Light-dependent reaction reveals three groups of substrate analogues with a fast, medium and slow reaction. To evaluate the kinetic data, the molar extinction coefficients in the reaction buffer had to be determined. At concentrations above 2 mole substrate/mole enzyme, inhibition was found for protochlorophyllide a and for the analogues.