Carbanionic
intermediates play a central role in the catalytic transformations
of amino acids performed by pyridoxal-5′-phosphate (PLP)-dependent
enzymes. Here, we make use of NMR crystallography—the synergistic
combination of solid-state nuclear magnetic resonance, X-ray crystallography,
and computational chemistry—to interrogate a carbanionic/quinonoid
intermediate analogue in the β-subunit active site of the PLP-requiring
enzyme tryptophan synthase. The solid-state NMR chemical shifts of
the PLP pyridine ring nitrogen and additional sites, coupled with
first-principles computational models, allow a detailed model of protonation
states for ionizable groups on the cofactor, substrates, and nearby
catalytic residues to be established. Most significantly, we find
that a deprotonated pyridine nitrogen on PLP precludes formation of
a true quinonoid species and that there is an equilibrium between
the phenolic and protonated Schiff base tautomeric forms of this intermediate.
Natural bond orbital analysis indicates that the latter builds up
negative charge at the substrate Cα and positive
charge at C4′ of the cofactor, consistent with its role as
the catalytic tautomer. These findings support the hypothesis that
the specificity for β-elimination/replacement versus transamination
is dictated in part by the protonation states of ionizable groups
on PLP and the reacting substrates and underscore the essential role
that NMR crystallography can play in characterizing both chemical
structure and dynamics within functioning enzyme active sites.
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.
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