Protonation States of the Tryptophan Synthase Internal Aldimine Active Site from Solid-State NMR Spectroscopy: Direct Observation of the Protonated Schiff Base Linkage to Pyridoxal-5鈥?Phosphate

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• 作者：Bethany G. Caulkins ; Baback Bastin ; Chen Yang ; Thomas J. Neubauer ; Robert P. Young ; Eduardo Hilario ; Yu-ming M. Huang ; Chia-en A. Chang ; Li Fan ; Michael F. Dunn ; Michael J. Marsella ; Leonard J. Mueller
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：September 17, 2014
• 年：2014
• 卷：136
• 期：37
• 页码：12824-12827
• 全文大小：255K
• ISSN：1520-5126

文摘

The acid鈥揵ase chemistry that drives catalysis in pyridoxal-5鈥?phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP鈥檚 role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine 蔚-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first ¹⁵N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase. Double-resonance experiments confirm the assignment of the Schiff base nitrogen, and additional ¹³C, ¹⁵N, and ³¹P chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established.