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′-Phosphate

• Published in: Journal of the American Chemical Society Vol. 136; no. 37; pp. 12824 - 12827
• Main Authors: Caulkins, Bethany G, Bastin, Baback, Yang, Chen, Neubauer, Thomas J, Young, Robert P, Hilario, Eduardo, Huang, Yu-ming M, Chang, Chia-en A, Fan, Li, Dunn, Michael F, Marsella, Michael J, Mueller, Leonard J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17.09.2014
• Subjects: active sites; Catalysis; catalytic activity; Catalytic Domain; Coenzymes; Communication; Enzymes; Linkages; Lysine; Models, Molecular; nitrogen; nuclear magnetic resonance spectroscopy; Nuclear Magnetic Resonance, Biomolecular; phosphorus; Protonation; Protons; pyridoxal phosphate; Pyridoxal Phosphate - chemistry; Pyridoxal Phosphate - metabolism; Salmonella typhimurium - chemistry; Salmonella typhimurium - enzymology; Salmonella typhimurium - metabolism; Schiff bases; Schiff Bases - chemistry; Schiff Bases - metabolism; stable isotopes; Tryptophan; tryptophan synthase; Tryptophan Synthase - chemistry; Tryptophan Synthase - metabolism
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/ja506267d

The acid–base chemistry that drives catalysis in pyridoxal-5′-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP’s 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 ε-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.