X-ray and NMR Crystallography in an Enzyme Active Site: The Indoline Quinonoid Intermediate in Tryptophan Synthase

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• 作者：Jinfeng Lai ; Dimitri Niks ; Yachong Wang ; Tatiana Domratcheva ; Thomas R. M. Barends ; Friedrich Schwarz ; Ryan A. Olsen ; Douglas W. Elliott ; M. Qaiser Fatmi ; Chia-en A. Chang ; Ilme Schlichting ; Michael F. Dunn ; Leonard J. Mueller
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：January 12, 2011
• 年：2011
• 卷：133
• 期：1
• 页码：4-7
• 全文大小：235K
• 年卷期：v.133,no.1(January 12, 2011)
• ISSN：1520-5126

文摘

Chemical-level details such as protonation and hybridization state are critical for understanding enzyme mechanism and function. Even at high resolution, these details are difficult to determine by X-ray crystallography alone. The chemical shift in NMR spectroscopy, however, is an extremely sensitive probe of the chemical environment, making solid-state NMR spectroscopy and X-ray crystallography a powerful combination for defining chemically detailed three-dimensional structures. Here we adopted this combined approach to determine the chemically rich crystal structure of the indoline quinonoid intermediate in the pyridoxal-5鈥?phosphate-dependent enzyme tryptophan synthase under conditions of active catalysis. Models of the active site were developed using a synergistic approach in which the structure of this reactive substrate analogue was optimized using ab initio computational chemistry in the presence of side-chain residues fixed at their crystallographically determined coordinates. Various models of charge and protonation state for the substrate and nearby catalytic residues could be uniquely distinguished by their calculated effects on the chemical shifts measured at specifically <sup>13sup>C- and <sup>15sup>N-labeled positions on the substrate. Our model suggests the importance of an equilibrium between tautomeric forms of the substrate, with the protonation state of the major isomer directing the next catalytic step.