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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Structural basis of catalysis by monometalated methionine aminopeptidase
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Structural basis of catalysis by monometalated methionine aminopeptidase

机译：单金属甲硫氨酸氨基肽酶催化的结构基础

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摘要

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 metallo-aminopeptidases, 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.
机译：蛋氨酸氨基肽酶（MetAP）从新合成的蛋白质中去除氨基末端的蛋氨酸残基，它是开发抗菌和抗癌药物的目标。 MetAP以及其他金属氨基肽酶的可用X射线结构显示了一个活性位点，该位点包含两个相邻的由水分子或氢氧根离子桥接的二价金属离子。双金属化结构的优势自然导致提出了涉及两种金属离子的催化机理。但是，动力学研究表明，在许多情况下，仅需一个金属离子即可发挥全部活性。通过限制晶体生长过程中存在的金属离子的数量，我们现在获得了大肠杆菌MetAP与正亮氨酸膦酸酯，过渡态类似物和仅在活性位点结合的单个Mn（II）离子的复合物的晶体结构。在表示为M1的位置上，以及相同配合物的三个相关结构显示出从单Mn（II）形式向二Mn（II）形式的转变。还解决了非配体结构。鉴于单金属化MetAP的全部动力学能力，与M1位点相比占据M2位点的结合常数弱得多以及新确定的结构，我们提出了一种通过大肠杆菌MetAP水解肽键水解的机制。我们还建议，在某些情况下，双金属形式的金属水解酶的结晶可能是误导性的实验假象，并且在生成结构时必须谨慎，以帮助阐明反应机理或支持结构辅助的药物设计工作。

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