14 research outputs found
Linear Free Energy Relationship Analysis of Transition State Mimicry by 3âDeoxyâd-<i>arabino-</i>heptulosonate-7-phosphate (DAHP) Oxime, a DAHP Synthase Inhibitor and Phosphate Mimic
3-Deoxy-d-<i>arabino-</i>heptulosonate-7-phosphate
(DAHP) synthase catalyzes an aldol-like reaction of phosphoenolpyruvate
(PEP) with erythrose 4-phosphate (E4P) to form DAHP in the first step
of the shikimate biosynthetic pathway. DAHP oxime, in which an oxime
replaces the ketone, is a potent inhibitor, with <i>K</i><sub>i</sub> = 1.5 ÎŒM. Linear free energy relationship (LFER)
analysis of DAHP oxime inhibition using DAHP synthase mutants revealed
an excellent correlation between transition state stabilization and
inhibition. The equations of LFER analysis were rederived to formalize
the possibility of proportional, rather than equal, changes in the
free energies of transition state stabilization and inhibitor binding,
in accord with the fact that the majority of LFER analyses in the
literature demonstrate nonunity slopes. A slope of unity, <i>m</i> = 1, indicates that catalysis and inhibitor binding are
equally sensitive to perturbations such as mutations or modified inhibitor/substrate
structures. Slopes <1 or >1 indicate that inhibitor binding
is
less sensitive or more sensitive, respectively, to perturbations than
is catalysis. LFER analysis using the tetramolecular specificity constant,
that is, plotting logÂ(<i>K</i><sub>M,Mn</sub><i>K</i><sub>M,PEP</sub><i>K</i><sub>M,E4P</sub>/<i>k</i><sub>cat</sub>) versus logÂ(<i>K</i><sub>i</sub>), revealed
a slope, <i>m</i>, of 0.34, with <i>r</i><sup>2</sup> = 0.93. This provides evidence that DAHP oxime is mimicking
the first irreversible transition state of the DAHP synthase reaction,
presumably phosphate departure from the tetrahedral intermediate.
This is evidence that the oxime group can act as a functional, as
well as structural, mimic of phosphate groups
Transition State Analysis of Enolpyruvylshikimate 3âPhosphate (EPSP) Synthase (AroA)-Catalyzed EPSP Hydrolysis
Proton transfer to carbon atoms is a significant catalytic
challenge
because of the large intrinsic energetic barrier and the frequently
unfavorable thermodynamics. The main catalytic challenge for enolpyruvylshikimate
3-phosphate synthase (EPSP synthase, AroA) is protonating the methylene
carbon atom of phosphoenolpyruvate, or EPSP, in the reverse reaction.
We performed transition state analysis using kinetic isotope effects
(KIEs) on AroA-catalyzed EPSP hydrolysis, which also begins with a
methylene carbon (C3) protonation, as an analog of AroAâs reverse
reaction. As part of this analysis, an inorganic phosphate scavenging
system was developed to remove phosphate which, though present in
microscopic amounts in solution, is ubiquitous. The reaction was stepwise,
with irreversible C3 protonation to form an EPSP cation intermediate;
that is, an A<sub>H</sub><sup>âĄ</sup>*A<sub>N</sub> mechanism.
The large experimental 3-<sup>14</sup>C KIE, 1.032 ± 0.005, indicated
strong coupling of C3 with the motion of the transferring proton.
Calculated 3-<sup>14</sup>C KIEs for computational transition state
models revealed that the transition state occurs early during C3âH<sup>+</sup> bond formation, with a C3âH<sup>+</sup> bond order
of â0.24. The observed solvent deuterium KIE, 0.97 ± 0.04,
was the lowest observed to date for this type of reaction, but consistent
with a very early transition state. The large 2-<sup>14</sup>C KIE
reflected an âelectrostatic sandwichâ formed by Asp313
and Glu341 to stabilize the positive charge at C2. In shifting the
transition state earlier than the acid-catalyzed reaction, AroA effected
a large Hammond shift, indicating that a significant part of AroAâs
catalytic strategy is to stabilize the positive charge in the EPSP
cation. A computational model containing all the charged amino acid
residues in the AroA active site close to the reactive center showed
a similar Hammond shift relative to the small transition state models
Potent Inhibition of 3âDeoxyâdâarabinoheptulosonate-7-phosphate (DAHP) Synthase by DAHP Oxime, a Phosphate Group Mimic
3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase
catalyzes the first step in the shikimate pathway. It catalyzes an
aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate
(E4P) to form DAHP. The kinetic mechanism was rapid equilibrium sequential
ordered ter ter, with the essential divalent metal ion, Mn<sup>2+</sup>, binding first, followed by PEP and E4P. DAHP oxime, in which an
oxime group replaces the keto oxygen, was a potent inhibitor, with <i>K</i><sub>i</sub> = 1.5 ± 0.4 ΌM, though with residual
activity at high inhibitor concentrations. It displayed slow-binding
inhibition with a residence time, <i>t</i><sub>R</sub>,
of 83 min. The crystal structure revealed that the oxime functional
group, combined with two crystallographic waters, bound at the same
location in the catalytic center as the phosphate group of the tetrahedral
intermediate. DAHP synthase has a dimer-of-dimers homotetrameric structure,
and DAHP oxime bound to only one subunit of each tight dimer. Inhibitor
binding was competitive with respect to all three substrates in the
subunits to which it bound. DAHP oxime did not overlap with the metal
binding site, so the cause of their mutually exclusive binding was
not clear. Similarly, there was no obvious structural reason for inhibitor
binding in only two subunits; however, changes in global hydrogen/deuterium
exchange showed large scale changes in protein dynamics upon inhibitor
binding. The <i>k</i><sub>cat</sub> value for the residual
activity at high inhibitor concentrations was 3-fold lower, and the
apparent <i>K</i><sub>M,E4P</sub> value decreased at least
10-fold. This positive cooperativity of binding between DAHP oxime
in subunits B and C, and E4P in subunits A and D appears to be the
dominant cause for incomplete inhibition at high inhibitor concentrations.
In spite of its lack of obvious structural similarity to phosphate,
the oxime and crystallographic waters acted as a small, neutral phosphate
mimic
Transition State Analysis of Acid-Catalyzed Hydrolysis of an Enol Ether, Enolpyruvylshikimate 3âPhosphate (EPSP)
Proton transfer to carbon represents a significant catalytic
challenge
because of the large intrinsic energetic barrier and the frequently
unfavorable thermodynamics. Multiple kinetic isotope effects (KIEs)
were measured for acid-catalyzed hydrolysis of the enol ether functionality
of enolpyruvylshikimate 3-phosphate (EPSP) as a nonenzymatic analog
of the EPSP synthase (AroA) reaction. The large solvent deuterium
KIE demonstrated that protonating C3 was the rate-limiting step, and
the lack of solvent hydron exchange into EPSP demonstrated that protonation
was irreversible. The reaction mechanism was stepwise, with C3, the
methylene carbon, being protonated to form a discrete oxacarbenium
ion intermediate before water attack at the cationic center, that
is, an A<sub>H</sub><sup>âĄ</sup>*A<sub>N</sub> (or A<sub>H</sub><sup>âĄ</sup> + A<sub>N</sub>) mechanism. The calculated 3-<sup>14</sup>C and 3,3-<sup>2</sup>H<sub>2</sub> KIEs varied as a function
of the extent of proton transfer at the transition state, as reflected
in the C3âH<sup>+</sup> bond order, <i>n</i><sub>C3âH+</sub>. The calculated 3-<sup>14</sup>C KIE was a function
primarily of C3 coupling with the movement of the transferring proton,
as reflected in the reaction coordinate contribution (<sup>light</sup>Îœ<sup>âĄ</sup>/<sup>heavy</sup>Îœ<sup>âĄ</sup>), rather than of changes in bonding. Coupling was strongest in early
and late transition states, where the reaction coordinate frequency
was lower. The other calculated <sup>14</sup>C and <sup>18</sup>O
KIEs were more sensitive to interactions with counterions and solvation
in the model structures than <i>n</i><sub>C3âH+</sub>. The KIEs revealed a moderately late transition state with significant
oxacarbenium ion character and with a C3âH<sup>+</sup> bond
order â0.6
A <sup>99m</sup>Tc-Labelled Tetrazine for Bioorthogonal Chemistry. Synthesis and Biodistribution Studies with Small Molecule <i>trans</i>-Cyclooctene Derivatives
<div><p>A convenient strategy to radiolabel a hydrazinonicotonic acid (HYNIC)-derived tetrazine with <sup>99m</sup>Tc was developed, and its utility for creating probes to image bone metabolism and bacterial infection using both active and pretargeting strategies was demonstrated. The <sup>99m</sup>Tc-labelled HYNIC-tetrazine was synthesized in 75% yield and exhibited high stability <i>in vitro</i> and <i>in vivo</i>. A <i>trans</i>-cyclooctene (TCO)-labelled bisphosphonate (TCO-BP) that binds to regions of active calcium metabolism was used to evaluate the utility of the labelled tetrazine for bioorthogonal chemistry. The pretargeting approach, with <sup>99m</sup>Tc-HYNIC-tetrazine administered to mice one hour after TCO-BP, showed significant uptake of radioactivity in regions of active bone metabolism (knees and shoulders) at 6 hours post-injection. For comparison, TCO-BP was reacted with <sup>99m</sup>Tc-HYNIC-tetrazine before injection and this active targeting also showed high specific uptake in the knees and shoulders, whereas control <sup>99m</sup>Tc-HYNIC-tetrazine alone did not. A TCO-vancomycin derivative was similarly employed for targeting <i>Staphylococcus aureus</i> infection <i>in vitro</i> and <i>in vivo</i>. Pretargeting and active targeting strategies showed 2.5- and 3-fold uptake, respectively, at the sites of a calf-muscle infection in a murine model, compared to the contralateral control muscle. These results demonstrate the utility of the <sup>99m</sup>Tc-HYNIC-tetrazine for preparing new technetium radiopharmaceuticals, including those based on small molecule targeting constructs containing TCO, using either active or pretargeting strategies.</p></div
Synthesis scheme for the preparation of 3.
<p>A protected form of HYNIC (<b>1</b>) was coupled to a commercially available tetrazine to form <b>2</b>. The Boc group was removed prior to labelling by treatment with TFA in DCM to produce <b>3</b>. Tz* = (4-(1,2,4,5-tetrazin-3-yl)phenyl) methanamine.</p
Synthesis scheme for <sup>99m</sup>Tc-HYNIC-tetrazine-TCO-vancomycin (7) from TCO-vancomycin (6) and 4.
<p>Synthesis scheme for <sup>99m</sup>Tc-HYNIC-tetrazine-TCO-vancomycin (7) from TCO-vancomycin (6) and 4.</p
Biodistribution data for 4.
<p>Data are presented as the mean (± SEM) percent injected dose per gram (%ID/g) for selected tissues and fluids from CD1 mice at 0.5, 1, 2 and 6 h post injection (n = 3 per time point). Approximately 0.88 MBq were administered per mouse. Full biodistribution data can be found in the supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167425#pone.0167425.s004" target="_blank">S4 File</a>).</p
Radiolabelling scheme for the HYNIC-tetrazine ligand 3 and HPLC chromatogram of the isolated product 4.
<p><sup>99m</sup>Tc labelling of the HYNIC-tetrazine <b>3</b> (A). A Îł-HPLC chromatogram of the purified final product <b>4</b> (B).</p
Biodistribution data for active targeting of <i>S</i>. <i>aureus</i> infection using <sup>99m</sup>Tc-HYNIC-tetrazine-TCO-vancomycin (7).
<p>Compounds <b>4</b> and <b>6</b> were combined prior to i.v. injection of Balb/c mice (n = 3 per time point). Select fluids and tissues were collected at 1 (gray bars) and 6 h (black bars) post injection, including the infected calf muscle (right), and the non-infected calf muscle (left). Data are expressed as the mean percent injected dose per gram (%ID/g) ± SEM. Tabulated biodistribution data can be found in the supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167425#pone.0167425.s004" target="_blank">S4 File</a>).</p