ATP-Dependent Carboxylation of Acetophenone by a Novel Type of Carboxylase
ATP-Dependent Carboxylation of Acetophenone by a Novel Type of Carboxylase
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DOI：
10.1128/jb.01423-09
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发表时间：
2010-03-01
影响因子：
3.2
通讯作者：
Heider, Johann
中科院分区：
文献类型：
--
作者：
Jobst, Bjoern;Schuehle, Karola;Heider, Johann
β-变形杆菌Aromatopharomaticum中的厌氧代谢是通过（S）-1-苯基乙醇厌氧氧化为苯乙酮而启动的。随后的苯乙酮羧化为苯甲酰乙酸是由苯乙酮诱导的酶催化的，该酶已被纯化和研究。相同的酶参与苯乙酮的代谢，在没有β-内酰胺的情况下。苯乙酮羧化酶由分子量为70、15、87、75和34 kDa的5个亚基组成，其基因（apcABCDE）形成表观操纵子。这种酶在生长在苯甲酸或苯乙酮上的细胞中以高水平合成，但在生长在苯甲酸盐上的细胞中不合成。在纯化过程中，苯乙酮羧化酶解离成分别由70-、15-、87-和75-kDa亚基（apcABCD基因产物）和34-kDa亚基（apcE基因产物）组成的无活性亚复合物。苯乙酮羧化酶活性恢复通过混合纯化的亚复合物。该酶含有1个Zn 2+离子/α β γ δ核心复合物，并且依赖于Mg 2+或Mn 2+的存在。尽管在酶中存在Zn，但它被Zn 2+离子强烈抑制。苯乙酮的羧化依赖于ATP水解为ADP和Pi，表现出每摩尔苯乙酮羧化2摩尔ATP的化学计量。在不存在第二底物的情况下，该酶显示与碳酸氢盐或苯乙酮解偶联的ATP酶活性。这些观察结果表明，这两种底物可能被磷酸化，这与氘代苯乙酮观察到的同位素交换活性和氨甲酰磷酸盐（一种羧基磷酸盐的结构类似物）的抑制作用一致。提出了苯乙酮羧化反应的可能机理。
Anaerobic ethylbenzene metabolism in the betaproteobacterium Aromatoleum aromaticum is initiated by anaerobic oxidation to acetophenone via (S)-1-phenylethanol. The subsequent carboxylation of acetophenone to benzoylacetate is catalyzed by an acetophenone-induced enzyme, which has been purified and studied. The same enzyme is involved in acetophenone metabolism in the absence of ethylbenzene. Acetophenone carboxylase consists of five subunits with molecular masses of 70, 15, 87, 75, and 34 kDa, whose genes (apcABCDE) form an apparent operon. The enzyme is synthesized at high levels in cells grown on ethylbenzene or acetophenone, but not in cells grown on benzoate. During purification, acetophenone carboxylase dissociates into inactive subcomplexes consisting of the 70-, 15-, 87-, and 75-kDa subunits (apcABCD gene products) and the 34-kDa subunit (apcE gene product), respectively. Acetophenone carboxylase activity was restored by mixing the purified subcomplexes. The enzyme contains 1 Zn2+ ion per alpha beta gamma delta core complex and is dependent on the presence of Mg2+ or Mn2+. In spite of the presence of Zn in the enzyme, it is strongly inhibited by Zn2+ ions. Carboxylation of acetophenone is dependent on ATP hydrolysis to ADP and Pi, exhibiting a stoichiometry of 2 mol ATP per mol acetophenone carboxylated. The enzyme shows uncoupled ATPase activity with either bicarbonate or acetophenone in the absence of the second substrate. These observations indicate that both substrates may be phosphorylated, which is consistent with isotope exchange activity observed with deuterated acetophenone and inhibition by carbamoylphosphate, a structural analogue of carboxyphosphate. A potential mechanism of ATP-dependent acetophenone carboxylation is suggested.