Ye Qi-Zhuang, Xie Sheng-Xue, Ma Ze-Qiang, Huang Min, Hanzlik Robert P
High Throughput Screening Laboratory and Department of Medicinal Chemistry, University of Kansas, 1501 Wakarusa Drive, Lawrence, KS 66045, USA.
Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9470-5. doi: 10.1073/pnas.0602433103. Epub 2006 Jun 12.
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.
甲硫氨酸氨肽酶（MetAP）可从新合成的蛋白质中去除氨基末端的甲硫氨酸残基，它是抗菌和抗癌药物开发的靶点。现有的MetAP以及其他金属氨肽酶的X射线结构显示，其活性位点含有两个相邻的二价金属离子，由一个水分子或氢氧根离子桥连。双金属结构的优势自然导致了涉及两个金属离子的催化机制的提出。然而，动力学研究表明，在许多情况下，完全活性仅需要单个金属离子。通过限制晶体生长过程中存在的金属离子量，我们现在获得了大肠杆菌MetAP与正亮氨酸膦酸酯（一种过渡态类似物）形成的复合物的晶体结构，在活性位点仅在指定为M1的位置结合了单个Mn（II）离子，以及同一复合物的三个相关结构，这些结构显示了从单Mn（II）形式到双Mn（II）形式的转变。还解析了一个无配体结构。鉴于单金属化MetAP具有完全的动力学活性，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，我们提出了大肠杆菌MetAP肽键水解的修正机制。我们还表明，在某些情况下，金属水解酶双金属化形式的结晶可能是一种误导性的实验假象，在生成结构以帮助阐明反应机制或支持基于结构的药物设计工作时必须谨慎。
