Towards a new interaction enzyme:coenzyme

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• 作者：Mart脙脙nez-J脙脙lvez ; Marta ; Tejero ; Jes脙脙s ; R. Peregrina ; Jos脙脙 ; Nogu脙脙s ; Isabel ; et. al.
• 关键词：Coenzyme specificity ; NAD(P)⁺/H ; Ferredoxin芒芒芒NADP⁺ reductase
• 刊名：Biophysical Chemistry
• 出版年：2005
• 期刊代码：11_03014622
• 类别：ch
• 出版时间：April 1, 2005
• 卷：115
• 期：2-3
• 页码：219-224
• 文件大小：134 K

摘要

Ferredoxin–NADP⁺ reductase catalyses NADP⁺ reduction, being specific for NADP⁺/H. To understand coenzyme specificity determinants and coenzyme specificity reversion, mutations at the NADP⁺/H pyrophosphate binding and of the C-terminal regions have been simultaneously introduced in Anabaena FNR. The T155G/A160T/L263P/Y303S mutant was produced. The mutated enzyme presents similar k_cat values for NADPH and NADH, around 2.5 times slower than that reported for WT FNR with NADPH. Its K_m value for NADH decreased 20-fold with regard to WT FNR, whereas the K_m for NADPH remains similar. The combined effect is a much higher catalytic efficiency for NAD⁺/H, with a minor decrease of that for NADP⁺/H. In the mutated enzyme, the specificity for NADPH versus NADH has been decreased from 67,500 times to only 12 times, being unable to discriminate between both coenzymes. Additionally, giving the role stated for the C-terminal Tyr in FNR, its role in the energetics of the FAD binding has been analysed.