1 research outputs found
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
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 <sup>15</sup>N 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 <sup>13</sup>C, <sup>15</sup>N, and <sup>31</sup>P chemical shift measurements of sites on the
PLP coenzyme allow a detailed model of coenzyme protonation states
to be established