Chemiluminescence of Mn2+-activated Rubisco:: temperature and pH responses differ between L2 and L8S8 forms, and inhibitors provide no evidence for involvement of active oxygen species
Chemiluminescence of Mn2+-activated Rubisco:: temperature and pH responses differ between L2 and L8S8 forms, and inhibitors provide no evidence for involvement of active oxygen species
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DOI：
10.1071/pp99032
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发表时间：
1999-01-01
期刊：
影响因子：
--
通讯作者：
Andrews, TJ
中科院分区：
文献类型：
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作者：
Cox, SD;Lilley, RM;Andrews, TJ
使用来自菠菜和聚球藻 PCC6301 的 L8S8 酶以及来自红色红螺菌的 L-2 酶研究了与 Mn2+ 激活的 D-核酮糖-1,5-二磷酸羧化酶/加氧酶 (Rubisco) 的加氧酶活性相关的化学发光。用光度计测量化学发光，用氧电极测量加氧酶活性。在 pH 8.1 的稳态下，菠菜 Rubisco 的相对发光率（每次加氧酶周转所发出的光）最高；来自聚球藻和红红藻的酶分别表现出大约菠菜值的一半和五分之一。 L8S8 酶的相对发光产量不受温度（20-40 摄氏度）和 pH（7.6-9.0）的影响。相比之下，R. rubrum Rubisco 的发光产量随温度和 pH 值变化，其 O-2 消耗活性表现出比 L8S8 酶更低的活化能。单线态 O-2 反应化合物二氮杂双环[2.2.2]辛烷和 10,10-二甲基-9,9-联吖啶二硝酸盐（光泽精）对化学发光没有影响。其他测试的化合物抑制化学发光，但也抑制 O-2 消耗。菠菜 Rubisco 化学发光的抑制需要几秒钟才能发挥其最大效果，这意味着抑制剂仅在催化循环的特定阶段才能到达活性位点。数据与作为发光源的活性位点的 Mn2+ 离子一致。
Chemiluminescence associated with the oxygenase activity of Mn2+-activated D-ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco) was investigated using the L8S8 enzymes from spinach and Synechococcus PCC6301, and the L-2 enzyme from Rhodospirillum rubrum. Chemiluminescence was measured with a luminometer and oxygenase activity with an oxygen electrode. The relative luminescence yield (light emitted per oxygenase turnover) in the steady-state at pH 8.1 was highest with spinach Rubisco; the enzymes from Synechococcus and R. rubrum exhibited approximately one half and one fifth, respectively, of the spinach value. The relative luminescence yield from the L8S8 enzymes was unaffected by temperature (20-40 degrees C) and pH (7.6-9.0). In contrast, the luminescence yield of R. rubrum Rubisco varied with temperature and pH, and its O-2-consuming activity exhibited a lower activation energy than that of the L8S8 enzymes. The singlet O-2-reactive compounds diazabicyclo[ 2.2.2] octane and 10,10-dimethyl-9,9-biacridinium dinitrate (lucigenin) had no effect on chemiluminescence. Other compounds tested inhibited chemiluminescence but also inhibited O-2 consumption. The inhibition of chemiluminescence of spinach Rubisco required several seconds to exert its maximal effect, implying that the inhibitors had access to the active site only at a particular stage of the catalytic cycle. The data are consistent with the Mn2+ ion at the active site being the source of the luminescence.