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 %26apos;-Phosphate

作者：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^*

来源：Journal of the American Chemical Society, 2014, 136(37): 12824-12827.

DOI：10.1021/ja506267d

The acid base chemistry that drives catalysis in pyridoxal-5%26apos;-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP%26apos;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 epsilon-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first N-15 NAIR 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 C-13, N-15, and P-31 chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established.

• 出版日期2014-9-17