Gencic Simonida, Grahame David A
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
J Biol Chem. 2003 Feb 21;278(8):6101-10. doi: 10.1074/jbc.M210484200. Epub 2002 Dec 2.
The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the central reaction of acetyl C-C bond cleavage in methanogens growing on acetate and is also responsible for synthesis of acetyl units during growth on C-1 substrates. The ACDS beta subunit contains nickel and an Fe/S center and reacts with acetyl-CoA forming an acetyl-enzyme intermediate presumably directly involved in acetyl C-C bond activation. To investigate the role of nickel in this process two forms of the Methanosarcina thermophila beta subunit were overexpressed in anaerobically grown Escherichia coli. Both contained an Fe/S center but lacked nickel and were inactive in acetyl-enzyme formation in redox-dependent acetyltransferase assays. However, high activity developed during incubation with NiCl(2). The native and nickel-reconstituted proteins both contained iron and nickel in a 2:1 ratio, with insignificant levels of other metals, including copper. Binding of nickel elicited marked changes in the UV-visible spectrum, with intense charge transfer bands indicating multiple thiolate ligation to nickel. The kinetics of nickel incorporation matched the time course for enzyme activation. Other divalent metal ions could not substitute for nickel in yielding catalytic activity. Acetyl-CoA was formed in reactions with CoA, CO, and methylcobalamin, directly demonstrating C-C bond activation by the beta subunit in the absence of other ACDS subunits. Nickel was indispensable in this process too and was needed to form a characteristic EPR-detectable enzyme-carbonyl adduct in reactions with CO. In contrast to enzyme activation, EPR signal formation did not require addition of reducing agent, indicating indirect catalytic involvement of the paramagnetic species. Site-directed mutagenesis indicated that Cys-278 and Cys-280 coordinate nickel, with Cys-189 essential for Fe/S cluster formation. The results are consistent with an Ni(2)[Fe(4)S(4)] arrangement at the active site. A mechanism for C-C bond activation is proposed that includes a specific role for the Fe(4)S(4) center and accounts for the absolute requirement for nickel.
乙酰辅酶A脱羰基酶/合酶（ACDS）复合物催化以乙酸盐为生长底物的产甲烷菌中乙酰基C-C键裂解的核心反应，并且在以C-1底物为生长底物时也负责乙酰基单元的合成。ACDSβ亚基含有镍和一个铁硫中心，并与乙酰辅酶A反应形成一种乙酰化酶中间体，推测该中间体直接参与乙酰基C-C键的活化。为了研究镍在此过程中的作用，两种嗜热嗜甲基菌β亚基在厌氧生长的大肠杆菌中过表达。两者都含有一个铁硫中心，但缺乏镍，并且在氧化还原依赖性乙酰转移酶测定中无活性形成乙酰化酶。然而，在与NiCl₂孵育期间产生了高活性。天然的和镍重构的蛋白质都含有铁和镍，其比例为2:1，其他金属（包括铜）的含量微不足道。镍的结合引起紫外可见光谱的显著变化，强烈的电荷转移带表明多个硫醇盐与镍配位。镍掺入的动力学与酶活化的时间进程相匹配。其他二价金属离子不能替代镍产生催化活性。在与辅酶A、CO和甲基钴胺素的反应中形成了乙酰辅酶A，直接证明了在没有其他ACDS亚基的情况下β亚基对C-C键的活化。镍在此过程中也是必不可少的，并且在与CO的反应中需要形成一种特征性的可通过电子顺磁共振检测到的酶-羰基加合物。与酶活化相反，电子顺磁共振信号形成不需要添加还原剂，表明顺磁物质间接参与催化。定点诱变表明，半胱氨酸-278和半胱氨酸-280与镍配位，半胱氨酸-189对铁硫簇的形成至关重要。结果与活性位点处的Ni(2)[Fe(4)S(4)]排列一致。提出了一种C-C键活化机制，其中包括铁硫中心的特定作用，并解释了对镍的绝对需求。
