Characterization of the acyl substrate binding pocket of acetyl-CoA synthetase
Characterization of the acyl substrate binding pocket of acetyl-CoA synthetase
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DOI：
10.1021/bi061023e
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发表时间：
2006-09-26
期刊：
影响因子：
2.9
通讯作者：
Smith, Kerry S.
中科院分区：
文献类型：
--
作者：
Ingram-Smith, Cheryl;Woods, Barrett I.;Smith, Kerry S.
AMP形成乙酰辅酶A合成酶[ACS；乙酸酯：辅酶A连接酶(AMP形成)，EC 6.2.1.1]催化乙酸乙酯在两步反应中活化为乙酰辅酶A。该酶是腺苷形成酶超家族的成员，包括萤火虫荧光素酶、非核糖体多肽合成酶以及酰基和芳基辅酶A合成酶/连接酶。尽管几个超家族成员的结构表明这些酶具有相似的折叠和结构域结构，但底物的低序列保守性和多样性限制了这些结构在理解这个超家族中更远亲缘的酶的底物结合方面的作用。肠炎沙门氏菌和酿酒酵母ACS1的晶体结构为研究ACS中的底物结合和催化作用提供了一种直接方法。在肠杆菌的ACS结构中，腺苷5‘-丙基磷酸的丙基位于疏水口袋中，它模拟了酰基腺苷的中间体。对嗜热甲烷杆菌Z245 ACS(MT-ACS1)在酿酒酵母ACS结构上的模拟显示了相似的活性部位结构，并且对已证实的ACSS的氨基酸序列进行了比对，表明组成推测的醋酸盐结合口袋的四个残基是很保守的。MT-ACS1的这四个残基，Ile(312)，Thr(313)，Val(388)和Trp(416)是被改变的目标，我们的结果支持他们确实形成了乙酸酯结合口袋，并且这些位置的改变显著改变了酶对乙酸酯的亲和力以及可以利用的酰基底物的范围。特别是，Trp416似乎是可在结合位点上调节的酰基链长度的主要决定因素。
AMP-forming acetyl-CoA synthetase [ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1] catalyzes the activation of acetate to acetyl-CoA in a two-step reaction. This enzyme is a member of the adenylate-forming enzyme superfamily that includes firefly luciferase, nonribosomal peptide synthetases, and acyl- and aryl-CoA synthetases/ligases. Although the structures of several superfamily members demonstrate that these enzymes have a similar fold and domain structure, the low sequence conservation and diversity of the substrates utilized have limited the utility of these structures in understanding substrate binding in more distantly related enzymes in this superfamily. The crystal structures of the Salmonella enterica ACS and Saccharomyces cerevisiae ACS1 have allowed a directed approach to investigating substrate binding and catalysis in ACS. In the S. enterica ACS structure, the propyl group of adenosine 5'-propylphosphate, which mimics the acyl-adenylate intermediate, lies in a hydrophobic pocket. Modeling of the Methanothermobacter thermautotrophicus Z245 ACS (MT-ACS1) on the S. cerevisiae ACS structure showed similar active site architecture, and alignment of the amino acid sequences of proven ACSs indicates that the four residues that compose the putative acetate binding pocket are well conserved. These four residues, Ile(312), Thr(313), Val(388), and Trp(416) of MT-ACS1, were targeted for alteration, and our results support that they do indeed form the acetate binding pocket and that alterations at these positions significantly alter the enzyme's affinity for acetate as well as the range of acyl substrates that can be utilized. In particular, Trp416 appears to be the primary determinant for acyl chain length that can be accommodated in the binding site.