Slow ligand-induced conformational switch increases the catalytic rate in Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase

Abstract

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 Km 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.

Structural and dynamical correlations in PfHGXPRT oligomers: A molecular dynamics simulation study

“…A compromised catalytic activity of this mutant enzyme suggested a correlation of loop X with the Leu-Lys peptide bond isomerization and dynamics of loop II. The isomerization of L76-K77 bond in loop I region is transmitted via a α-helix to loop X, and the same was discussed by us in our earlier report (Roy et al, 2015); it is further elaborated (Figure 5(b)) from a dynamical perspective here. However, herein we restrict our discussion to the ligand-free and ligand-bound states and do not consider the cis-trans isomerization of the aforementioned dipeptide and its associated events.…”

“…Point mutation of residues present in this loop (loop I) in human HGPRT (Balendiran et al, 1999) and T. cruzi HPRT (Canyuk et al, 2004, Butterworth et al, 2004 leads to significant changes at the interface, which in turn alters the catalytic efficiency of the corresponding enzymes. Due to the large barrier for this bond rotation (∼20-25 kcal/mol), the conformation of this dipeptide remains unchanged as seen from unbiased MD simulations (Roy et al, 2015). The dynamics of loop I in each of the subunits of the oligomers are summarized in Figure S5(a) of SI.…”

“…In the cis conformation of the dipeptide, the Lys 77 side chain moves away from the active site to provide space for the substrate and/or product molecules. However, in the free protein, the side chain of Lys 77 occupies the active site cavity in the trans conformation of the dipeptide where it interacts with Glu 144 through a salt-bridge (Roy et al, 2015). Point mutation of residues present in this loop (loop I) in human HGPRT (Balendiran et al, 1999) and T. cruzi HPRT (Canyuk et al, 2004, Butterworth et al, 2004 leads to significant changes at the interface, which in turn alters the catalytic efficiency of the corresponding enzymes.…”

Role of W181 in modulating kinetic properties of Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase

“…To determine the K m value for xanthine with unactivated W181 mutants, xanthine concentration was varied from 5 μ M to 200 μ M for W181S, and 10 μ M to 150 μ M for W181Y and W181F, while PRPP was fixed at 3 m M . Activation was carried out by incubating 30 μ M enzyme with 60 μ M IMP and 5 m M DTT in 10 m M potassium phosphate, pH 7.0 at 0 ̊C for 3 h . With IMP activated W181 mutants, K m value for PRPP was determined by varying PRPP concentrations from 0.1 m M to 2 m M for W181S, 0.05 m M to 1.5 m M for W181Y and 0.2 m M to 3 m M for W181F and keeping xanthine fixed at 100 μ M .…”

“…The catalytically competent form of PfHGXPRT is a tetramer. It exhibits an equilibrium between homo dimer and homo tetramer that is modulated by the presence of the substrate, phosphoribosylpyrophosphate (PRPP) and buffer conditions . The structure of a single subunit of the tetramer is shown in Figure .…”