Martínez-Júlvez M, Hurley J K, Tollin G, Gómez-Moreno C, Fillat M F
Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Spain.
Biochim Biophys Acta. 1996 Oct 17;1297(2):200-6. doi: 10.1016/s0167-4838(96)00024-6.
The complete petH gene product from Anabaena PCC 7119 has been overexpressed in E. coli and purified in order to determine the influence of the N-terminal extension on the interaction of ferredoxin-NADP+ reductase with its substrates. The intact 49 kDa FNR can be easily purified in a two-step procedure using batch extraction with DEAE-cellulose followed by Cibacron blue-Sepharose chromatography of the proteins unbound to DEAE. Isoelectric focusing of FNR shows several forms, with the major band at pH 6.26. The presence of the N-terminal extension increases the K(m) of FNR for NADPH by 4-fold and by 16.4-fold in the reduction reactions of DCPIP and cytochrome c. However, the K(m) for ferredoxin is 12-fold lower in the reaction catalyzed by the 49 kDa FNR than with the 36 kDa protein. This indicates that the presence of the third domain favours the interaction of FNR with ferredoxin, possibly due to the more positive net charge of the N-terminal extension. Comparable rate constants for both enzymes, were obtained for the photoreduction of NADP+ using photosynthetic membranes and also using rapid kinetic techniques. Slightly different ionic strength dependences of the rate constants were obtained, nevertheless, for both forms of the enzyme. These are a consequence of the structural differences that the proteins show at the N-terminal and of their effect on the interaction with ferredoxin.
为了确定N端延伸对铁氧化还原蛋白-NADP⁺还原酶与其底物相互作用的影响，已在大肠杆菌中对鱼腥藻PCC 7119完整的petH基因产物进行了过表达并纯化。完整的49 kDa FNR可以通过两步法轻松纯化，首先用DEAE-纤维素进行批量提取，然后对未结合到DEAE的蛋白质进行Cibacron blue-Sepharose层析。FNR的等电聚焦显示有几种形式，主要条带在pH 6.26处。在DCPIP和细胞色素c的还原反应中，N端延伸的存在使FNR对NADPH的K(m)增加了4倍和16.4倍。然而，在由49 kDa FNR催化的反应中，铁氧化还原蛋白的K(m)比36 kDa蛋白低12倍。这表明第三个结构域的存在有利于FNR与铁氧化还原蛋白的相互作用，这可能是由于N端延伸的净电荷更正。使用光合膜以及快速动力学技术对NADP⁺进行光还原时，两种酶获得了可比的速率常数。不过，对于两种形式的酶，速率常数的离子强度依赖性略有不同。这些是蛋白质在N端显示的结构差异及其对与铁氧化还原蛋白相互作用的影响的结果。
