44 research outputs found
Plasmodium falciparum Parasites Are Killed by a Transition State Analogue of Purine Nucleoside Phosphorylase in a Primate Animal Model
Plasmodium falciparum causes most of the one million annual deaths from malaria. Drug resistance is widespread and novel agents against new targets are needed to support combination-therapy approaches promoted by the World Health Organization. Plasmodium species are purine auxotrophs. Blocking purine nucleoside phosphorylase (PNP) kills cultured parasites by purine starvation. DADMe-Immucillin-G (BCX4945) is a transition state analogue of human and Plasmodium PNPs, binding with picomolar affinity. Here, we test BCX4945 in Aotus primates, an animal model for Plasmodium falciparum infections. Oral administration of BCX4945 for seven days results in parasite clearance and recrudescence in otherwise lethal infections of P. falciparum in Aotus monkeys. The molecular action of BCX4945 is demonstrated in crystal structures of human and P. falciparum PNPs. Metabolite analysis demonstrates that PNP blockade inhibits purine salvage and polyamine synthesis in the parasites. The efficacy, oral availability, chemical stability, unique mechanism of action and low toxicity of BCX4945 demonstrate potential for combination therapies with this novel antimalarial agent
Study protocol for the PHANTOM study: prehospital assessment of noninvasive tissue oximetry monitoring
Ribocation Transition State Capture and Rebound in Human Purine Nucleoside Phosphorylase
SummaryPurine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to the corresponding purine base and α-D-ribose 1-phosphate. Its genetic loss causes a lethal T cell deficiency. The highly reactive ribocation transition state of human PNP is protected from solvent by hydrophobic residues that sequester the catalytic site. The catalytic site was enlarged by replacing individual catalytic site amino acids with glycine. Reactivity of the ribocation transition state was tested for capture by water and other nucleophiles. In the absence of phosphate, inosine is hydrolyzed by native, Y88G, F159G, H257G, and F200G enzymes. Phosphorolysis but not hydrolysis is detected when phosphate is bound. An unprecedented N9-to-N3 isomerization of inosine is catalyzed by H257G and F200G in the presence of phosphate and by all PNPs in the absence of phosphate. These results establish a ribocation lifetime too short to permit capture by water. An enlarged catalytic site permits ribocation formation with relaxed geometric constraints, permitting nucleophilic rebound and N3-inosine isomerization
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
Arsenate and phosphate as nucleophiles at the transition states of human purine nucleoside phosphorylase
Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of 6-oxypurine (2′-deoxy)ribonucleosides, generating (2-deoxy)ribose 1-phosphate and the purine base. Transition-state models for inosine cleavage have been proposed with bovine, human, and malarial PNPs using arsenate as the nucleophile, since kinetic isotope effects (KIEs) are obscured on phosphorolysis due to high commitment factors. The Phe200Gly mutant of human PNP has a low forward and reverse commitment factors in the phosphorolytic reaction, permitting the measurement of competitive intrinsic KIEs on both arsenolysis and phosphorolysis of inosine. The intrinsic 1′-(14)C, 1′-(3)H, 2′-(2)H, 9-(15)N, and 5′-(3)H(2) KIEs for inosine were measured for arsenolysis and phosphorolysis. Except for the remote 5′-(3)H(2), and some slight difference between the 2′-(2)H KIEs, all isotope effects originating in the reaction coordinate are the same within experimental error. Hence, arsenolysis and phosphorolysis proceed through closely related transition states. Although electrostatically similar, the volume of arsenate is greater than phosphate and supports a steric influence to explain the differences in the 5′-(3)H(2) KIEs. Density functional theory calculations provide quantitative models of the transition states for Phe200Gly human PNP-catalyzed arsenolysis and phosphorolysis, selected upon matching calculated and experimental KIEs. The models confirm the striking resemblance between the transition states for the two reactions