Boll M, Albracht S S, Fuchs G
Mikrobiologie, Institut Biologie II, Universität Freiburg, Germany.
Eur J Biochem. 1997 Mar 15;244(3):840-51. doi: 10.1111/j.1432-1033.1997.00840.x.
An enzyme was recently described, benzoyl-CoA reductase (dearomatizing), which catalyses the ATP-driven reduction of the aromatic ring of benzoyl-CoA yielding a non-aromatic CoA thioester, ADP and phosphate [Boll, M. & Fuchs, G. (1995) Eur. J. Biochem. 234, 921-933]. The 170-kDa enzyme consists of four different subunits and contains approximately 12 Fe and acid-labile sulfur/mol. Benzoyl-CoA reductase exhibits ATPase activity in the absence of substrate. It is shown that only the reduced form of this iron-sulfur protein has ATPase activity. ATPase activity is reversibly lost when the enzyme is oxidized by thionine; reduction of the enzyme fully restores ATPase and ring-reduction activity. 2 mol ATP are hydrolyzed/2 mol electrons transferred in the course of the reaction. The product ADP acts as competitive inhibitor (Ki = 1.1 mM) for ATP in benzoyl-CoA reduction; ADP inhibits ATPase activity to the same extent as ring-reduction activity. EPR investigation of the dithionite-reduced enzyme suggested the presence of two separate [2Fe-2S] clusters and two interacting [4Fe-4S] clusters. Addition of MgATP to the reduced enzyme resulted in a new isotropic signal at g = 5.15 and a weak signal at g = 12; in controls with MgADP only a minor signal at g = 5.15 was observed. The positions, shapes and temperature dependencies of these MgATP-induced signals are indicative for excited states of a S = 7/2 spin multiplet. The [2Fe-2S] signals were not affected by ATP, but one of the [4Fe-4S] clusters became slowly oxidized. Addition of both benzoyl-CoA and MgATP resulted in a major oxidation of the iron-sulfur clusters accompanied by the appearance of some minor signals of unknown origin in the g = 2.037-1.96 region. Neither the benzoyl-CoA plus MgATP-oxidized nor the thionine-oxidized enzyme showed the ATP-dependent formation of the high-spin signals of the reduced enzyme. At present we hypothesize that the S = 7/2 signal is due to an ATP-induced change of one of the [4Fe-4S] clusters. The data suggest that hydrolysis of MgATP is required to activate the enzyme; in the absence of substrate the energy involved in this activation dissipates. MgATP-driven formation of this excited state of the reduced enzyme rather than transfer of electrons from the reduced enzyme to the aromatic substrate appears to be the rate-limiting step in the catalytic cycle. We suggest that the excited state is required to overcome the high activation energy associated with the loss of the aromatic character and/or to render ring reduction irreversible.
最近发现了一种酶，即苯甲酰辅酶A还原酶（脱芳香化），它催化由ATP驱动的苯甲酰辅酶A芳香环的还原反应，生成一种非芳香性的辅酶A硫酯、ADP和磷酸[博尔，M. & 富克斯，G.（1995年）《欧洲生物化学杂志》234卷，921 - 933页]。这种170 kDa的酶由四个不同的亚基组成，每摩尔含有约12个铁原子和对酸不稳定的硫原子。苯甲酰辅酶A还原酶在没有底物时表现出ATP酶活性。结果表明，只有这种铁硫蛋白的还原形式具有ATP酶活性。当该酶被硫堇氧化时，ATP酶活性可逆丧失；酶的还原完全恢复了ATP酶和环还原活性。在反应过程中，每转移2摩尔电子会水解2摩尔ATP。产物ADP在苯甲酰辅酶A还原反应中作为ATP的竞争性抑制剂（Ki = 1.1 mM）；ADP对ATP酶活性的抑制程度与对环还原活性的抑制程度相同。对连二亚硫酸盐还原的酶进行的电子顺磁共振研究表明存在两个独立的[2Fe - 2S]簇和两个相互作用的[4Fe - 4S]簇。向还原的酶中添加MgATP会在g = 5.15处产生一个新的各向同性信号，并在g = 12处产生一个微弱信号；在仅添加MgADP的对照实验中，仅在g = 5.15处观察到一个微弱信号。这些MgATP诱导信号的位置、形状和温度依赖性表明是S = 7/2自旋多重态的激发态。[2Fe - 2S]信号不受ATP影响，但其中一个[4Fe - 4S]簇会缓慢氧化。同时添加苯甲酰辅酶A和MgATP会导致铁硫簇发生主要氧化，并在g = 2.037 - 1.96区域出现一些未知来源的微弱信号。无论是苯甲酰辅酶A加MgATP氧化的酶还是硫堇氧化的酶，都未显示出还原酶的高自旋信号的ATP依赖性形成。目前我们推测S = 7/2信号是由于[4Fe - 4S]簇之一的ATP诱导变化所致。数据表明，MgATP的水解是激活该酶所必需的；在没有底物的情况下，这种激活过程中涉及的能量会消散。MgATP驱动还原酶形成这种激发态，而不是还原酶向芳香底物的电子转移，似乎是催化循环中的限速步骤。我们认为，这种激发态是为了克服与芳香性丧失相关的高活化能和/或使环还原不可逆。
