7 research outputs found
Pre-Steady-State Kinetic Analysis of 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase from <i>Mycobacterium tuberculosis</i> Reveals Partially Rate-Limiting Product Release by Parallel Pathways
As part of the non-mevalonate pathway for the biosynthesis
of the
isoprenoid precursor isopentenyl pyrophosphate, 1-deoxy-d-xylulose-5-phosphate (DXP) reductoisomerase (DXR) catalyzes the
conversion of DXP into 2-<i>C</i>-methyl-d-erythritol
4-phosphate (MEP) by consecutive isomerization and NADPH-dependent
reduction reactions. Because this pathway is essential to many infectious
organisms but is absent in humans, DXR is a target for drug discovery.
In an attempt to characterize its kinetic mechanism and identify rate-limiting
steps, we present the first complete transient kinetic investigation
of DXR. Stopped-flow fluorescence measurements with <i>Mycobacterium
tuberculosis</i> DXR (<i>Mt</i>DXR) revealed that NADPH
and MEP bind to the free enzyme and that the two bind together to
generate a nonproductive ternary complex. Unlike the <i>Escherichia
coli</i> orthologue, <i>Mt</i>DXR exhibited a burst
in the oxidation of NADPH during pre-steady-state reactions, indicating
a partially rate-limiting step follows chemistry. By monitoring NADPH
fluorescence during these experiments, the transient generation of <i>Mt</i>DXR·NADPH·MEP was observed. Global kinetic analysis
supports a model involving random substrate binding and ordered release
of NADP<sup>+</sup> followed by MEP. The partially rate-limiting release
of MEP occurs via two pathwaysî—¸directly from the binary complex
and indirectly via the <i>Mt</i>DXR·NADPH·MEP
complexî—¸the partitioning being dependent on NADPH concentration.
Previous mechanistic studies, including kinetic isotope effects and
product inhibition, are discussed in light of this kinetic mechanism
DXP Reductoisomerase: Reaction of the Substrate in Pieces Reveals a Catalytic Role for the Nonreacting Phosphodianion Group
The
role of the nonreacting phosphodianion group of 1-deoxy-d-xylulose-5-phosphate (DXP) in catalysis by DXP reductoisomerase
(DXR) was investigated for the reaction of the “substrate in
pieces”. The truncated substrate 1-deoxy-l-erythrulose
is converted by DXR to 2-<i>C</i>-methylglycerol with a <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> that is 10<sup>6</sup>-fold lower than that for DXP. Phosphite dianion was found
to be a nonessential activator, providing 3.2 kcal/mol of transition
state stabilization for the truncated substrate. These results implicate
a phosphate-driven conformational change involving loop closure over
the DXR active site to generate an environment poised for catalysis
Cysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3‑Nitropropionate
Isocitrate lyase (ICL) catalyzes
the reversible cleavage of isocitrate
into succinate and glyoxylate. It is the first committed step in the
glyoxylate cycle used by some organisms, including <i>Mycobacterium
tuberculosis</i>, where it has been shown to be essential for
cell survival during chronic infection. The pH–rate and pD–rate
profiles measured in the direction of isocitrate synthesis revealed
solvent kinetic isotope effects (KIEs) of 1.7 ± 0.4 for <sup>D<sub>2</sub>O</sup><i>V</i> and 0.56 ± 0.07 for <sup>D<sub>2</sub>O</sup>(<i>V</i>/<i>K</i><sub>succinate</sub>). Whereas the <sup>D<sub>2</sub>O</sup><i>V</i> is consistent
with partially rate-limiting proton transfer during formation of the
hydroxyl group of isocitrate, the large inverse <sup>D<sub>2</sub>O</sup>(<i>V</i>/<i>K</i><sub>succinate</sub>) indicates that substantially different kinetic parameters exist
when the enzyme is saturated with succinate. Inhibition by 3-nitropropionate
(3-NP), a succinate analogue, was found to proceed through an unusual
double slow-onset process featuring formation of a complex with a <i>K</i><sub>i</sub> of 3.3 ± 0.2 μM during the first
minute, followed by formation of a final complex with a <i>K</i><sub>i</sub>* of 44 ± 10 nM over the course of several minutes
to hours. Stopped-flow measurements during the first minute revealed
an apparent solvent KIE of 0.40 ± 0.03 for association and unity
for dissociation. In contrast, itaconate, a succinate analogue lacking
an acidic α-proton, did not display slow-binding behavior and
yielded a <sup>D<sub>2</sub>O</sup><i>K</i><sub>i</sub> of
1.0 ± 0.2. These results support a common mechanism for catalysis
with succinate and inhibition by 3-NP featuring (1) an unfavorable
prebinding isomerization of the active site Cys191–His193 pair
to the thiolate–imidazolium form, a process that is favored
in D<sub>2</sub>O, and (2) the transfer of a proton from succinate
or 3-NP to Cys191. These findings also indicate that propionate-3-nitronate,
which is the conjugate base of 3-NP and the “true inhibitor”
of ICL, does not bind directly and must be generated enzymatically
The Nitro Group as a Masked Electrophile in Covalent Enzyme Inhibition
We report the unprecedented
reaction between a nitroalkane and
an active-site cysteine residue to yield a thiohydroximate adduct.
Structural and kinetic evidence suggests the nitro group is activated
by conversion to its nitronic acid tautomer within the active site.
The nitro group, therefore, shows promise as a masked electrophile
in the design of covalent inhibitors targeting binding pockets with
appropriately placed cysteine and general acid residues
The Role of Phosphate in a Multistep Enzymatic Reaction: Reactions of the Substrate and Intermediate in Pieces
Several mechanistically unrelated
enzymes utilize the binding energy
of their substrate’s nonreacting phosphoryl group to accelerate
catalysis. Evidence for the involvement of the phosphodianion in transition
state formation has come from reactions of the substrate in pieces,
in which reaction of a truncated substrate lacking its phosphorylmethyl
group is activated by inorganic phosphite. What has remained unknown
until now is how the phosphodianion group influences the reaction
energetics at different points along the reaction coordinate. 1-Deoxy-d-xylulose-5-phosphate (DXP) reductoisomerase (DXR), which catalyzes
the isomerization of DXP to 2-<i>C</i>-methyl-d-erythrose 4-phosphate (MEsP) and subsequent NADPH-dependent reduction,
presents a unique opportunity to address this concern. Previously,
we have reported the effect of covalently linked phosphate on the
energetics of DXP turnover. Through the use of chemically synthesized
MEsP and its phosphate-truncated analogue, 2-<i>C</i>-methyl-d-glyceraldehyde, the current study revealed a loss of 6.1 kcal/mol
of kinetic barrier stabilization upon truncation, of which 4.4 kcal/mol
was regained in the presence of phosphite dianion. The activating
effect of phosphite was accompanied by apparent tightening of its
interactions within the active site at the intermediate stage of the
reaction, suggesting a role of the phosphodianion in disfavoring intermediate
release and in modulation of the on-enzyme isomerization equilibrium.
The results of kinetic isotope effect and structural studies indicate
rate limitation by physical steps when the covalent linkage is severed.
These striking differences in the energetics of the natural reaction
and the reactions in pieces provide a deeper insight into the contribution
of enzyme–phosphodianion interactions to the reaction coordinate
Highly Precise Measurement of Kinetic Isotope Effects Using <sup>1</sup>H‑Detected 2D [<sup>13</sup>C,<sup>1</sup>H]-HSQC NMR Spectroscopy
A new method is presented for measuring kinetic isotope
effects
(KIEs) by <sup>1</sup>H-detected 2D [<sup>13</sup>C,<sup>1</sup>H]-heteronuclear
single quantum coherence (HSQC) NMR spectroscopy. The high accuracy
of this approach was exemplified for the reaction catalyzed by glucose-6-phosphate
dehydrogenase by comparing the 1-<sup>13</sup>C KIE with the published
value obtained using isotope ratio mass spectrometry. High precision
was demonstrated for the reaction catalyzed by 1-deoxy-d-xylulose-5-phosphate
reductoisomerase from Mycobacterium tuberculosis. 2-, 3-, and 4-<sup>13</sup>C KIEs were found to be 1.0031(4), 1.0303(12),
and 1.0148(2), respectively. These KIEs provide evidence for a cleanly
rate-limiting retroaldol step during isomerization. The high intrinsic
sensitivity and signal dispersion of 2D [<sup>13</sup>C,<sup>1</sup>H]-HSQC offer new avenues to study challenging systems where low
substrate concentration and/or signal overlap impedes 1D <sup>13</sup>C NMR data acquisition. Moreover, this approach can take advantage
of highest-field spectrometers, which are commonly equipped for <sup>1</sup>H detection with cryogenic probes
Alteration of the Flexible Loop in 1‑Deoxy‑d‑xylulose-5-phosphate Reductoisomerase Boosts Enthalpy-Driven Inhibition by Fosmidomycin
1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR),
which catalyzes the first committed step in the 2-<i>C</i>-methyl-d-erythritol 4-phosphate pathway of isoprenoid biosynthesis
used by <i>Mycobacterium tuberculosis</i> and other infectious
microorganisms, is absent in humans and therefore an attractive drug
target. Fosmidomycin is a nanomolar inhibitor of DXR, but despite
great efforts, few analogues with comparable potency have been developed.
DXR contains a strictly conserved residue, Trp203, within a flexible
loop that closes over and interacts with the bound inhibitor. We report
that while mutation to Ala or Gly abolishes activity, mutation to
Phe and Tyr only modestly impacts <i>k</i><sub>cat</sub> and <i>K</i><sub>m</sub>. Moreover, pre-steady-state kinetics
and primary deuterium kinetic isotope effects indicate that while
turnover is largely limited by product release for the wild-type enzyme,
chemistry is significantly more rate-limiting for W203F and W203Y.
Surprisingly, these mutants are more sensitive to inhibition by fosmidomycin,
resulting in <i>K</i><sub>m</sub>/<i>K</i><sub>i</sub> ratios up to 19-fold higher than that of wild-type DXR. In
agreement, isothermal titration calorimetry revealed that fosmidomycin
binds up to 11-fold more tightly to these mutants. Most strikingly,
mutation strongly tips the entropy–enthalpy balance of total
binding energy from 50% to 75% and 91% enthalpy in W203F and W203Y,
respectively. X-ray crystal structures suggest that these enthalpy
differences may be linked to differences in hydrogen bond interactions
involving a water network connecting fosmidomycin’s phosphonate
group to the protein. These results confirm the importance of the
flexible loop, in particular Trp203, in ligand binding and suggest
that improved inhibitor affinity may be obtained against the wild-type
protein by introducing interactions with this loop and/or the surrounding
structured water network