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.
Sulfamate groups (NHSO(3)(-)) are important structural elements in the glycosaminoglycans (GAGs) heparin and heparan sulfate (HS). In this work, proton nuclear magnetic resonance (NMR) line-shape analysis is used to explore the solvent exchange properties of the sulfamate NH groups within heparin-related mono-, di-, tetra- and pentasaccharides as a function of pH and temperature. The results of these experiments identified a persistent hydrogen bond within the Arixtra (fondaparinux sodium) pentasaccharide between the internal glucosamine sulfamate NH and the adjacent 3-O-sulfo group. This discovery provides new insights into the solution structure of the Arixtra pentasaccharide and suggests that 3-O-sulfation of the heparin N-sulfoglucosamine (GlcNS) residues pre-organize the secondary structure in a way that facilitates binding to antithrombin-III. NMR studies of the GlcNS NH groups can provide important information about heparin structure complementary to that available from NMR spectral analysis of the carbon-bound protons.
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.
Alkanes and [B12X12]2− (X = Cl, Br) are both stable compounds which are difficult to functionalize. Here we demonstrate the formation of a boron−carbon bond between these substances in a two-step process. Fragmentation of [B12X12]2− in the gas phase generates highly reactive [B12X11]− ions which spontaneously react with alkanes. The reaction mechanism was investigated using tandem mass spectrometry and gas-phase vibrational spectroscopy combined with electronic structure calculations. [B12X11]− reacts by an electrophilic substitution of a proton in an alkane resulting in a B−C bond formation. The product is a dianionic [B12X11CnH2n+1]2− species, to which H+ is electrostatically bound. High-flux ion soft landing was performed to codeposit [B12X11]− and complex organic molecules (phthalates) in thin layers on surfaces. Molecular structure analysis of the product films revealed that C−H functionalization by [B12X11]− occurred in the presence of other more reactive functional groups. This observation demonstrates the utility of highly reactive fragment ions for selective bond formation processes and may pave the way for the use of gas-phase ion chemistry for the generation of complex molecular structures in the condensed phase.
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