Kinetic mechanism of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase

Roy, Sourav; Nagappa, Lakshmeesha Kempaiah; Prahladarao, Vasudeva S.; Balaram, Hemalatha

doi:10.1016/j.molbiopara.2016.02.006

Cited by 15 publications

(30 citation statements)

References 38 publications

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“…Catalytic site residues that make contact with the reactants are identical. Kinetic mechanisms for the human, 8 schistosomal, 9 Trypanosoma cruzi , 10 and more recently P. falciparum 11 enzymes are reported to be ordered bi-bi substrate addition and product release, in which PRPP binds first to the enzyme, followed by 6-oxopurine binding. PP i dissociates first from the enzyme, followed by nucleotide release.…”

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confidence: 99%

Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Plasmodium falciparum

2017

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Plasmodium falciparum 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 phosphor-ibosyltransferase (HGXPRT) catalyzes the freely reversible Mg2+-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 (PfHGXPRT) is essential as hypoxanthine is the major precursor in purine metabolism. We used specific heavy atom labels in PRPP and hypoxanthine to measure primary (1-14C and 9-15N) and secondary (1-3H and 7-15N) intrinsic kinetic isotope effect (KIE) values for PfHGXPRT. Intrinsic isotope effects contain information for understanding enzymatic transition state properties. The transition state of PfHGXPRT was explored by matching KIE values predicted from quantum mechanical calculations to the intrinsic values determined experimentally. This approach provides information about PfHGXPRT transition state bond lengths, geometry, and atomic charge distribution. The transition state structure of PfHGXPRT 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 PfHGXPRT forms nucleotides through a well-developed and near-symmetrical DN*AN, SN1-like transition state.

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confidence: 99%

Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Plasmodium falciparum

2017

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“…PfHGXPRT has been shown to exhibit hysteresis, where the time course of the enzyme catalyzed reaction exhibits a pronounced lag whose duration is enzyme and PRPP concentration dependent . Similar to the wild type enzyme, the W181 mutants also displayed hysteretic behaviour with the lag duration decreasing with increase in enzyme concentration.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2016

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Hypoxanthine-guanine-xanthine phosphoribosyltransference (HGXPRT), a key enzyme in the purine salvage pathway of the malarial parasite, Plasmodium falciparum (Pf), catalyses the conversion of hypoxanthine, guanine, and xanthine to their corresponding mononucleotides; IMP, GMP, and XMP, respectively. Out of the five active site loops (I, II, III, III', and IV) in PfHGXPRT, loop III' facilitates the closure of the hood over the core domain which is the penultimate step during enzymatic catalysis. PfHGXPRT mutants were constructed wherein Trp 181 in loop III' was substituted with Ser, Thr, Tyr, and Phe. The mutants (W181S, W181Y and W181F), when examined for xanthine phosphoribosylation activity, showed an increase in K for PRPP by 2.1-3.4 fold under unactivated condition and a decrease in catalytic efficiency by more than 5-fold under activated condition as compared to that of the wild-type enzyme. The W181T mutant showed 10-fold reduced xanthine phosphoribosylation activity. Furthermore, molecular dynamics simulations of WT and in silico W181S/Y/F/T PfHGXPRT mutants bound to IMP.PPi.Mg have been carried out to address the effect of the mutation of W181 on the overall dynamics of the systems and identify local changes in loop III'. Dynamic cross-correlation analyses show a communication between loop III' and the substrate binding site. Differential cross-correlation maps indicate altered communication among different regions in the mutants. Changes in the local contacts and hydrogen bonding between residue 181 with the nearby residues cause altered substrate affinity and catalytic efficiency of the mutant enzymes. Proteins 2016; 84:1658-1669. © 2016 Wiley Periodicals, Inc.

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“…In this study, strong binding properties were observed between Pf HGXPRT and the ligands, this is characterised by the good splitting observed; the lowest KD (1.5272 × 10 −12 ) and highest KD confidence (±2.193 × 10 −9 ). The increased binding capabilities Pf HGXPRT can be attributed to the formation of new cis-and trans-conformational change resulting in better interaction and peptide bonds formed in the binding site between the ligand and the receptor [7]. The observed data indicate that conformational changes in the Pf HGXPRT hydration shell, charge and surface area which alter mobility are induced by the MA and UAA.…”

Section: Mst Binding Analysis Of Ursolic Acid Acetate and Iso-mukaadimentioning

confidence: 89%

“…Several techniques have been developed in an effort to try and better understand protein-protein and protein-ligand interaction, as the basis of protein interaction opens the doorway to understanding vital reactions in organisms [14]. The study of proteins binding to ligands requires that we study and understand how proteins interact with drugs in terms of their conformation, binding affinities, kinetics and thermodynamics, in an effort to develop specific drugs that target proteins that have a vital role in the diseases and illness [7,14]. UV-Vis spectrophotometry can be used to monitor protein secondary structure changes through three aromatic amino acids: Phe, Tyr and Trp.…”

Section: Uv-vis Spectrometer Analysismentioning

confidence: 99%

“…In this study, we investigated Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (Pf HGXPRT), an essential malaria protein, as a potential target for iso-mukaadial acetate and ursolic acid acetate. The Plasmodium falciparum parasite lacks the necessary de novo pathway enzymes to create its own purines and hence is dependent upon the salvage pathway [6][7][8]. Thus this difference can be exploited to design novel drugs to inhibit Pf HGXPRT [9][10][11].…”

Section: Introductionmentioning

confidence: 99%

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Evaluating Iso-Mukaadial Acetate and Ursolic Acid Acetate as Plasmodium falciparum Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase Inhibitors

et al. 2019

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To date, Plasmodium falciparum is one of the most lethal strains of the malaria parasite. P. falciparum lacks the required enzymes to create its own purines via the de novo pathway, thereby making Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPT) a crucial enzyme in the malaria life cycle. Recently, studies have described iso-mukaadial acetate and ursolic acid acetate as promising antimalarials. However, the mode of action is still unknown, thus, the current study sought to investigate the selective inhibitory and binding actions of iso-mukaadial acetate and ursolic acid acetate against recombinant PfHGXPT using in-silico and experimental approaches. Recombinant PfHGXPT protein was expressed using E. coli BL21 cells and homogeneously purified by affinity chromatography. Experimentally, iso-mukaadial acetate and ursolic acid acetate, respectively, demonstrated direct inhibitory activity towards PfHGXPT in a dose-dependent manner. The binding affinity of iso-mukaadial acetate and ursolic acid acetate on the PfHGXPT dissociation constant (KD), where it was found that 0.0833 µM and 2.8396 µM, respectively, are indicative of strong binding. The mode of action for the observed antimalarial activity was further established by a molecular docking study. The molecular docking and dynamics simulations show specific interactions and high affinity within the binding pocket of Plasmodium falciparum and human hypoxanthine-guanine phosphoribosyl transferases. The predicted in silico absorption, distribution, metabolism and excretion/toxicity (ADME/T) properties predicted that the iso-mukaadial acetate ligand may follow the criteria for orally active drugs. The theoretical calculation derived from ADME, molecular docking and dynamics provide in-depth information into the structural basis, specific bonding and non-bonding interactions governing the inhibition of malarial. Taken together, these findings provide a basis for the recommendation of iso-mukaadial acetate and ursolic acid acetate as high-affinity ligands and drug candidates against PfHGXPT.

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Kinetic mechanism of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase

Cited by 15 publications

References 38 publications

Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Plasmodium falciparum

Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Plasmodium falciparum

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

Evaluating Iso-Mukaadial Acetate and Ursolic Acid Acetate as Plasmodium falciparum Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase Inhibitors

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