Aliverti A, Piubelli L, Zanetti G, Lübberstedt T, Herrmann R G, Curti B
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Biochemistry. 1993 Jun 29;32(25):6374-80. doi: 10.1021/bi00076a010.
To investigate the functional role of the cysteine residues present in the spinach ferredoxin-NADP+ oxidoreductase, we individually replaced each of the five cysteine residues with serine using site-directed mutagenesis. All of the mutant reductases were correctly assembled in Escherichia coli except for the C42S mutant protein. C114S and C137S mutant enzymes apparently showed structural and kinetic properties very similar to those of the wild-type reductase. However, C272S and C132S mutations yielded enzymes with a decreased catalytic activity in the ferredoxin-dependent reaction (14 and 31% of the wild type, respectively). Whereas the C132S was fully competent in the diaphorase reaction, the C272S mutant flavoprotein showed a 35-fold reduction in catalytic efficiency with respect to the wild-type enzyme (0.4 versus 14.28 microM-1 s-1) due to a substantial decrease of kcat. NADP+ binding by the C272S mutant enzyme was apparently quantitatively the same (Kd = 37 microM) but qualitatively different, as shown by the differential spectrum. Stopped-flow experiments showed that the enzyme-FAD reduction rate was considerably decreased in the C272S mutant reductase, along with a much lower yield of the charge-transfer transient species. It is inferred from these data that the charge transfer (FAD-NADPH) between the reductase and NADPH is required for hydride transfer from the pyridine nucleotide to flavin to occur with a rate compatible with catalysis.
为了研究菠菜铁氧化还原蛋白 - NADP⁺氧化还原酶中半胱氨酸残基的功能作用，我们使用定点诱变技术将五个半胱氨酸残基中的每一个分别替换为丝氨酸。除了C42S突变蛋白外，所有突变型还原酶都在大肠杆菌中正确组装。C114S和C137S突变酶的结构和动力学性质显然与野生型还原酶非常相似。然而，C272S和C132S突变产生的酶在铁氧化还原蛋白依赖性反应中的催化活性降低（分别为野生型的14%和31%）。虽然C132S在双氢酶反应中完全有活性，但C272S突变黄素蛋白相对于野生型酶的催化效率降低了35倍（分别为0.4和14.28 μM⁻¹ s⁻¹），这是由于kcat大幅降低。如差示光谱所示，C272S突变酶与NADP⁺的结合在数量上显然相同（Kd = 37 μM），但在质量上有所不同。停流实验表明，C272S突变还原酶中酶 - FAD的还原速率显著降低，同时电荷转移瞬态物种的产率也低得多。从这些数据可以推断，还原酶与NADPH之间的电荷转移（FAD - NADPH）是使氢化物从吡啶核苷酸转移到黄素以与催化相适应的速率发生所必需的。
