Aliverti A, Corrado M E, Zanetti G
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
FEBS Lett. 1994 May 2;343(3):247-50. doi: 10.1016/0014-5793(94)80565-2.
A mutant of spinach ferredoxin-NADP+ reductase, in which Lys-88 has been changed to glutamine, has been obtained by site-directed mutagenesis. The mutant enzyme was fully active as a diaphorase, but partially impaired in ferredoxin-dependent cytochrome c reductase activity. By steady-state kinetics, the Km for ferredoxin of the K88Q enzyme was found to have increased 10-fold, whereas the kcat was unaffected by the amino acid replacement. The interaction between oxidized ferredoxin and the enzyme forms was also studied by spectrofluorimetric titration: Kd values of 110 and 10 nM were determined for the mutant and wild-type proteins, respectively. These data point out the importance of a positive charge at position 88 of the reductase for the interaction with ferredoxin, confirming previous cross-linking studies.
通过定点诱变获得了菠菜铁氧化还原蛋白 - NADP⁺还原酶的一个突变体，其中第88位赖氨酸已被替换为谷氨酰胺。该突变酶作为一种黄递酶具有完全活性，但在依赖铁氧化还原蛋白的细胞色素c还原酶活性方面部分受损。通过稳态动力学研究发现，K88Q酶对铁氧化还原蛋白的Km值增加了10倍，而kcat不受氨基酸替换的影响。还通过荧光光谱滴定法研究了氧化态铁氧化还原蛋白与酶形式之间的相互作用：分别测定了突变体和野生型蛋白的解离常数Kd值为110 nM和10 nM。这些数据指出了还原酶第88位正电荷对于与铁氧化还原蛋白相互作用的重要性，证实了先前的交联研究。
