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Solution‐State 17 O Quadrupole Central‐Transition NMR Spectroscopy in the Active Site of Tryptophan Synthase
Author(s) -
Young Robert P.,
Caulkins Bethany G.,
Borchardt Dan,
Bulloch Daryl N.,
Larive Cynthia K.,
Dunn Michael F.,
Mueller Leonard J.
Publication year - 2016
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201508898
Subject(s) - chemistry , active site , indoline , nuclear magnetic resonance spectroscopy , substrate (aquarium) , tryptophan synthase , stereochemistry , photochemistry , catalysis , tryptophan , organic chemistry , amino acid , biochemistry , oceanography , geology
Oxygen is an essential participant in the acid–base chemistry that takes place within many enzyme active sites, yet has remained virtually silent as a probe in NMR spectroscopy. Here, we demonstrate the first use of solution‐state 17 O quadrupole central‐transition NMR spectroscopy to characterize enzymatic intermediates under conditions of active catalysis. In the 143 kDa pyridoxal‐5′‐phosphate‐dependent enzyme tryptophan synthase, reactions of the α‐aminoacrylate intermediate with the nucleophiles indoline and 2‐aminophenol correlate with an upfield shift of the substrate carboxylate oxygen resonances. First principles calculations suggest that the increased shieldings for these quinonoid intermediates result from the net increase in the charge density of the substrate–cofactor π‐bonding network, particularly at the adjacent α‐carbon site.