12 research outputs found
Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from <i>Plasmodium falciparum</i>
<i>Plasmodium
falciparum</i> parasites are purine auxotrophs that rely exclusively
on the salvage of preformed purines from their human hosts to supply
the requirement for purine nucleotides. Hypoxanthine-guanine-xanthine
phosphoribosyltransferase (HGXPRT) catalyzes the freely reversible
Mg<sup>2+</sup>-dependent conversion of 6-oxopurine bases to their
respective nucleotides and inorganic pyrophosphate. The phosphoribosyl
group is derived from 5-phospho-α-d-ribosyl 1-pyrophosphate
(PRPP). The enzyme from malaria parasites (<i>Pf</i>HGXPRT)
is essential as hypoxanthine is the major precursor in purine metabolism.
We used specific heavy atom labels in PRPP and hypoxanthine to measure
primary (1-<sup>14</sup>C and 9-<sup>15</sup>N) and secondary (1-<sup>3</sup>H and 7-<sup>15</sup>N) intrinsic kinetic isotope effect (KIE)
values for <i>Pf</i>HGXPRT. Intrinsic isotope effects contain
information for understanding enzymatic transition state properties.
The transition state of <i>Pf</i>HGXPRT was explored by
matching KIE values predicted from quantum mechanical calculations
to the intrinsic values determined experimentally. This approach provides
information about <i>Pf</i>HGXPRT transition state bond
lengths, geometry, and atomic charge distribution. The transition
state structure of <i>Pf</i>HGXPRT was determined in the
physiological direction of addition of ribose 5-phosphate to hypoxanthine
by overcoming the chemical instability of PRPP. The transition state
for <i>Pf</i>HGXPRT forms nucleotides through a well-developed
and near-symmetrical D<sub>N</sub>*A<sub>N</sub>, S<sub>N</sub>1-like
transition state
The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy
Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-l-phosphate (R1P). The human UP type 1 (hUP1) is a molecular target for the design of inhibitors intended to boost endogenous uridine levels to rescue normal tissues from the toxicity of fluoropyrimidine nucleoside chemotherapeutic agents, such as capecitabine and 5-fluorouracil. Here, we describe a method to obtain homogeneous recombinant hUP1, and present initial velocity, product inhibition, and equilibrium binding data. These results suggest that hUP1 catalyzes uridine phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate binds first followed by the binding of uridine, and uracil dissociates first, followed by RIP release. Fluorescence titration at equilibrium showed cooperative binding of either Pi or RIP binding to hUP1. Amino acid residues involved in either catalysis or substrate binding were proposed based on pH-rate profiles. (C) 2010 Elsevier B.V. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq
Biochemical characterization of recombinant guaA-encoded guanosine monophosphate synthetase (EC 6.3.5.2) from Mycobacterium tuberculosis H37Rv strain
Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum
Crystal structure and molecular dynamics studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis associated with acyclovir
Synthesis of <i>bis</i>-Phosphate Iminoaltritol Enantiomers and Structural Characterization with Adenine Phosphoribosyltransferase
Phosphoribosyl
transferases (PRTs) are essential in nucleotide
synthesis and salvage, amino acid, and vitamin synthesis. Transition
state analysis of several PRTs has demonstrated ribocation-like transition
states with a partial positive charge residing on the pentose ring.
Core chemistry for synthesis of transition state analogues related
to the 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP)
reactant of these enzymes could be developed by stereospecific placement
of <i>bis</i>-phosphate groups on an iminoaltritol ring.
Cationic character is provided by the imino group and the <i>bis</i>-phosphates anchor both the 1- and 5-phosphate binding
sites. We provide a facile synthetic path to these molecules. Cyclic-nitrone
redox methodology was applied to the stereocontrolled synthesis of
three stereoisomers of a selectively monoprotected diol relevant to
the synthesis of transition-state analogue inhibitors. These polyhydroxylated
pyrrolidine natural product analogues were <i>bis</i>-phosphorylated
to generate analogues of the ribocationic form of 5-phosphoribosyl
1-phosphate. A safe, high yielding synthesis of the key intermediate
represents a new route to these transition state mimics. An enantiomeric
pair of iminoaltritol <i>bis</i>-phosphates (L-DIAB and
D-DIAB) was prepared and shown to display inhibition of <i>Plasmodium
falciparum</i> orotate phosphoribosyltransferase and <i>Saccharomyces cerevisiae</i> adenine phosphoribosyltransferase
(ScAPRT). Crystallographic inhibitor binding analysis of L- and D-DIAB
bound to the catalytic sites of ScAPRT demonstrates accommodation
of both enantiomers by altered ring geometry and <i>bis</i>-phosphate catalytic site contacts
Continuous Fluorescence Assays for Reactions Involving Adenine
5′-Methylthioadenosine
phosphorylase (MTAP) and 5′-methylthioadenosine
nucleosidase (MTAN) catalyze the phosphorolysis and hydrolysis of
5′-methylthioadenosine (MTA), respectively. Both enzymes have
low <i>K</i><sub>M</sub> values for their substrates. Kinetic
assays for these enzymes are challenging, as the ultraviolet absorbance
spectra for reactant MTA and product adenine are similar. We report
a new assay using 2-amino-5′-methylthioadenosine (2AMTA) as
an alternative substrate for MTAP and MTAN enzymes. Hydrolysis or
phosphorolysis of 2AMTA forms 2,6-diaminopurine, a fluorescent and
easily quantitated product. We kinetically characterize 2AMTA with
human MTAP, bacterial MTANs and use 2,6-diaminopurine as a fluorescent
substrate for yeast adenine phosphoribosyltransferase. 2AMTA was used
as the substrate to kinetically characterize the dissociation constants
for three-transition-state analogue inhibitors of MTAP and MTAN. Kinetic
values obtained from continuous fluorescent assays with MTA were in
good agreement with previously measured literature values, but gave
smaller experimental errors. Chemical synthesis from ribose and 2,6-dichloropurine
provided crystalline 2AMTA as the oxalate salt. Chemo-enzymatic synthesis
from ribose and 2,6-diaminopurine produced 2-amino-<i>S</i>-adenosylmethionine for hydrolytic conversion to 2AMTA. Interaction
of 2AMTA with human MTAP was also characterized by pre-steady-state
kinetics and by analysis of the crystal structure in a complex with
sulfate as a catalytically inert analogue of phosphate. This assay
is suitable for inhibitor screening by detection of fluorescent product,
for quantitative analysis of hits by rapid and accurate measurement
of inhibition constants in continuous assays, and pre-steady-state
kinetic analysis of the target enzymes
Transition-state inhibitors of purine salvage and other prospective enzyme targets in malaria
Molecular, kinetic, thermodynamic, and structural analyses of Mycobacterium tuberculosis hisD-encoded metal-dependent dimeric histidinol dehydrogenase (EC 1.1.1.23)
The emergence of drug-resistant strains of Mycobacterium tuberculosis, the major causative agent of tuberculosis (TB), and the deadly HIV-TB co-infection have led to an urgent need for the development of new anti-TB drugs. The histidine biosynthetic pathway is present in bacteria, archaebacteria, lower eukaryotes and plants, but is absent in mammals. Disruption of the hisD gene has been shown to be essential for M. tuberculosis survival. Here we present cloning, expression and purification of recombinant hisD-encoded histidinol dehydrogenase (MtHisD). N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtHisD. Analytical gel filtration, metal requirement analysis, steady-state kinetics and isothermal titration calorimetry data showed that homodimeric MtHisD is a metalloprotein that follows a Bi Uni Uni Bi Ping-Pong mechanism. pH-rate profiles and a three-dimensional model of MtHisD allowed proposal of amino acid residues involved in either catalysis or substrate(s) binding. (C) 2011 Elsevier B.V. All rights reserved
Discovery of new inhibitors of Mycobacterium tuberculosis InhA enzyme using virtual screening and a 3D-pharmacophore-based approach
Mycobacterium tuberculosis InhA (MtInhA) is an attractive enzyme to drug discovery efforts due to its validation as an effective biological target for tuberculosis therapy. In this work, two different virtual-ligand-screening approaches were applied in order to identify new InhA inhibitors' candidates from a library of ligands selected from the ZINC database. First, a 3-D pharmacophore model was built based on 36 available MtInhA crystal structures. By combining structure-based and ligand-based information, four pharmacophoric points were designed to select molecules able to satisfy the binding features of MtInhA substrate-binding cavity. The second approach consisted of using four well established docking programs, with different search algorithms, to compare the binding mode and score of the selected molecules from the aforementioned library. After detailed analyses of the results, six ligands were selected for in vitro analysis. Three of these molecules presented a satisfactory inhibitory activity with IC50 values ranging from 24 (±2) μM to 83 (±5) μM. The best compound presented an uncompetitive inhibition mode to NADH and 2-trans-dodecenoyl-CoA substrates, with Ki values of 24 (±3) μM and 20 (±2) μM, respectively. These molecules were not yet described as antituberculars or as InhA inhibitors, making its novelty interesting to start efforts on ligand optimization in order to identify new effective drugs against tuberculosis having InhA as a target. More studies are underway to dissect the discovered uncompetitive inhibitor interactions with MtInhA.CNPq (559917/2010, 305984/2012-8, 520182/99-5, 304051/1975-06)Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB)MCTI / CNPqDECIT / MSFAPERGS / CNPq / PRONEX (2009)CAPE