Slow ligand-induced conformational switch increases the catalytic rate in Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferaseElectronic supplementary information (ESI) available: The supporting information contains sections describing the methods and outcomes of construction of W181S and W181S/F197W, purification of PfHGXPRT and mutants (Sections S1 and S2), solvent kinetic isotope effects (Section S3), isothermal titration calorimetry (Section S4) and conformational ch
P. falciparum (Pf) hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) exhibits a unique mechanism of activation where the enzyme switches from a low activity (unactivated) to a high activity (activated) state upon pre-incubation with substrate/products. Xanthine phosphoribosylation by...
Saved in:
| Main Authors | , , , , , |
|---|---|
| Format | Journal Article |
| Language | English |
| Published |
21.04.2015
|
| Online Access | Get full text |
Cover
Loading…
| Summary: | P. falciparum
(Pf) hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) exhibits a unique mechanism of activation where the enzyme switches from a low activity (unactivated) to a high activity (activated) state upon pre-incubation with substrate/products. Xanthine phosphoribosylation by unactivated PfHGXPRT exhibits a lag phase, the duration of which reduces with an increase in concentration of the enzyme or substrate, PRPP·Mg
2+
. Activated PfHGXPRT does not display the lag phase and exhibits a ten-fold drop in the
K
m
value for PRPP·Mg
2+
. These observations suggest the involvement of ligand-mediated oligomerization and conformational changes in the process of activation. The dipeptide Leu-Lys in the PPi binding site of human and
T. gondii
HG(X)PRT that facilitates PRPP·Mg
2+
binding by isomerization from
trans
to
cis
conformation is conserved in PfHGXPRT. Free energy calculations using the well-tempered metadynamics technique show the ligand-free enzyme to be more stable when this dipeptide is in the
trans
conformation than in the
cis
conformation. The high rotational energy barrier observed for the conformational change from experimental and computational studies permits delineation of the activation mechanism.
Coupled events of ligand-induced isomerization and oligomerization in catalysis by PfHGXPRT. |
|---|---|
| Bibliography: | the dipeptide omega dihedral angle (Cα-C-N-Cα), a comparison of Ramachandran plots of free PfHGXPRT and free human HGPRT enzymes, the conformational changes of PfHGXPRT during isomerization of the Leu76-Lys77 peptide bond and the tetramer structure of PfHGXPRT with assignment of the individual subunits are provided. Web enhanced: two web enhanced objects are available in the online version of the paper. This includes two movies in mpeg format that highlight: (1) the opening of loop II, and (2) the conformational change of the Leu76-Lys77 peptide bond. See DOI and apo structure has been included in Section S6. Additionally, data on the hysteretic behavior of PfHGXPRT in potassium phosphate, the oligomeric state of PfHGXPRT in Tris HCl and potassium phosphate buffers, the increase of rate constants with increasing concentration of PfHGXPRT and PRPP, the temperature dependence of lag duration, the relative magnitudes of activation in potassium phosphate and Tris HCl, a comparison of the biochemical properties of PfHGXPRT mutants, the change of fluorescence emission properties of W181S/F197W with an increase in IMP concentration, an estimation of the dissociation constant of the PfHGXPRT·IMP complex using isothermal titration calorimetry and the dependence of the lag duration on PRPP concentration observed using a stopped flow spectrophotometer, the dihedral angle change of the Leu-Lys dipeptide in unbiased MD simulations, gas phase and solvent phase energy barrier calculations, free energy profiles obtained from four different WTM simulations for rotation about the Leu-Lys peptide bond in the ligand-free PfHGXPRT enzyme, the Lys77 side chain interaction with Glu144 and Ile146, the distance between the nitrogen atom (Nξ) of the Lys77 side chain and the carbon atom (Cδ) of the Glu144 side chain Electronic supplementary information (ESI) available: The supporting information contains sections describing the methods and outcomes of construction of W181S and W181S/F197W, purification of PfHGXPRT and mutants (Sections S1 and S2), solvent kinetic isotope effects (Section S3), isothermal titration calorimetry (Section S4) and conformational changes (Section S5) in the dipeptide in the gas phase and in water. A comparison between the 10.1039/c5mb00136f cis versus trans |
| ISSN: | 1742-206X 1742-2051 |
| DOI: | 10.1039/c5mb00136f |