Lennon B W, Williams C H
Department of Biological Chemistry, University of Michigan, Ann Arbor, 48105, USA.
Biochemistry. 1996 Apr 16;35(15):4704-12. doi: 10.1021/bi952521i.
Thioredoxin reductase from Escherichia coli is a member of the pyridine nucleotide-disulfide oxidoreductase family, and contains one FAD and one redox-active disulfide per subunit. It is known that two other well-studied members of this family, lipoamide dehydrogenase and glutathione reductase, cycle between the two electron-reduced and fully oxidized forms in catalysis. Enzyme-monitored turnover shows that the spectrum of thioredoxin reductase during turnover represents fully reduced flavin with NADP(H) bound. Whether the pyridine nucleotide bound is NADPH or NADP+ is dependent on the concentration of each species, i.e., how far turnover has progressed. It is also shown that the midpoint potentials of this enzyme are increased through the differential binding of NADP+ to the oxidized and reduced form of the enzyme. When combined with other kinetic and oxidation/reduction studies of this enzyme, these results indicate that thioredoxin reductase cycles between the four-electron-reduced and two-electron-reduced forms in catalysis, and that it does so with pyridine nucleotide bound. These results clarify the mechanism of thioredoxin reductase in relation to the known structure the enzyme, and provide support for earlier work in which we proposed that this enzyme utilizes a ternary complex mechanism in catalysis.
来自大肠杆菌的硫氧还蛋白还原酶是吡啶核苷酸 - 二硫化物氧化还原酶家族的成员，每个亚基含有一个FAD和一个氧化还原活性二硫键。已知该家族另外两个经过充分研究的成员，硫辛酰胺脱氢酶和谷胱甘肽还原酶，在催化过程中在双电子还原和完全氧化形式之间循环。酶监测的周转表明，周转过程中硫氧还蛋白还原酶的光谱代表结合了NADP（H）的完全还原的黄素。结合的吡啶核苷酸是NADPH还是NADP +取决于每种物质的浓度，即周转进行的程度。还表明，通过NADP +与酶的氧化形式和还原形式的差异结合，该酶的中点电位会增加。当与该酶的其他动力学和氧化/还原研究相结合时，这些结果表明硫氧还蛋白还原酶在催化过程中在四电子还原和双电子还原形式之间循环，并且在结合吡啶核苷酸的情况下进行循环。这些结果阐明了硫氧还蛋白还原酶与已知酶结构相关的机制，并为我们早期提出该酶在催化中利用三元复合物机制的工作提供了支持。
