Structural basis of catalysis by monometalated methionine aminopeptidase.
Structural basis of catalysis by monometalated methionine aminopeptidase.
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DOI：
10.1073/pnas.0602433103
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发表时间：
2006-06
影响因子：
11.1
通讯作者：
Q. Ye;S. Xie;Ze-Qiang Ma;Min Huang;R. Hanzlik
中科院分区：
文献类型：
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作者：
Q. Ye;S. Xie;Ze-Qiang Ma;Min Huang;R. Hanzlik
甲硫氨酸氨基肽酶（MetAP）是新合成的蛋白质中去除氨基末端甲硫氨酸残基的酶，是抗菌和抗癌药物开发的靶点。可用的MetAP以及其他金属氨基肽酶的X射线结构显示含有通过水分子或氢氧根离子桥接的两个相邻的二价金属离子的活性位点。双金属化结构的优势自然导致提出涉及两种金属离子的催化机制。然而，动力学研究表明，在许多情况下，只需要一个单一的金属离子的全部活动。通过限制晶体生长过程中存在的金属离子的量，我们现在已经获得了大肠杆菌MetAP与正亮氨酸膦酸盐，过渡态类似物的复合物的晶体结构，并且仅在指定M1的位置中的活性位点处结合单个Mn（II）离子，以及显示从单Mn（II）形式到二Mn（II）形式的过渡的相同复合物的三个相关结构。一个无配体的结构也得到了解决。考虑到单取代的MetAP的完全动力学能力，与M1位点相比，M2位点占据的结合常数要弱得多，以及新确定的结构，我们提出了E. coli MetAP。我们还建议，金属水解酶的二金属化形式的结晶，在某些情况下，可能是一个误导性的实验文物，必须谨慎时，产生的结构，以帮助阐明反应机制或支持结构辅助药物设计的努力。
Methionine aminopeptidase (MetAP) removes the amino-terminal methionine residue from newly synthesized proteins, and it is a target for the development of antibacterial and anticancer agents. Available x-ray structures of MetAP, as well as other metalloaminopeptidases, show an active site containing two adjacent divalent metal ions bridged by a water molecule or hydroxide ion. The predominance of dimetalated structures leads naturally to proposed mechanisms of catalysis involving both metal ions. However, kinetic studies indicate that in many cases, only a single metal ion is required for full activity. By limiting the amount of metal ion present during crystal growth, we have now obtained a crystal structure for a complex of Escherichia coli MetAP with norleucine phosphonate, a transition-state analog, and only a single Mn(II) ion bound at the active site in the position designated M1, and three related structures of the same complex that show the transition from the mono-Mn(II) form to the di-Mn(II) form. An unliganded structure was also solved. In view of the full kinetic competence of the monometalated MetAP, the much weaker binding constant for occupancy of the M2 site compared with the M1 site, and the newly determined structures, we propose a revised mechanism of peptide bond hydrolysis by E. coli MetAP. We also suggest that the crystallization of dimetalated forms of metallohydrolases may, in some cases, be a misleading experimental artifact, and caution must be taken when structures are generated to aid in elucidation of reaction mechanisms or to support structure-aided drug design efforts.