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

Abstract

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.

Inhibition and Mechanism of Plasmodium falciparum Hypoxanthine–Guanine–Xanthine Phosphoribosyltransferase

“…Pf HGXPRT from Escherichia coli extracts copurifies with substoichiometric amounts of purine and may account for the results obtained by activation with PRPP as a single agent. 25,26,32,35 Activated Pf HGXPRT gave K m values for hypoxanthine and PRPP of 10 ± 1 and 185 ± 14 μM, respectively, with a k cat of 0.12 ± 0.01 s −1 . Kinetic assays with unactivated Pf HGXPRT gave K m values for hypoxanthine and PRPP of 2.7 ± 0.3 and 55 ± 11 μM, respectively, and a k cat of 0.020 ± 0.002 s −1 .…”

“…ImmHP is a transition state analogue of Pf HGXPRT, and its activation of the enzyme captures the enzyme in a configuration near the transition state, consistent with stabilizing the active tetrameric form of the enzyme (Figure E). Because ImmHP is a powerful inhibitor of Pf HGXPRT, its slow release from the enzyme makes it incompatible as an activating agent. , Earlier reports for Pf HGXPRT activation involved incubation of (1) 30 μM Pf HGXPRT with 60 μM IMP, (2) 1 mM PRPP and 12 mM MgCl 2 , in 5 mM DTT and 10 mM phosphate buffer pH 7.0 at 0 °C for 3 h, or (3) 1 mM MgCl 2 , 1 mM pyrophosphate, and 5 mM DTT in 10 mM phosphate buffer pH 7.0 at 0 °C for 3 h. ,, In our hands, these protocols provided an equilibrium of monomeric, dimeric, and tetrameric species in the cross-linking analysis (Figure ). Pf HGXPRT from Escherichia coli extracts copurifies with substoichiometric amounts of purine and may account for the results obtained by activation with PRPP as a single agent. ,,, …”

“…Pf HGXPRT was activated by preincubating 30 μM Pf HGXPRT with 1 mM hypoxanthine, 2 mM PRPP, 10 mM MgCl 2 , 1 mM DTT, and 50 mM potassium phosphate pH 7.4 in a 50 μL reaction mix for 30 min at 25 °C. Activation by magnesium pyrophosphate, IMP, and PRPP was performed according to the method outlined by Roy et al ,, A 60-fold dilution of the activation mix was made into assay mixtures described below to determine the efficiency of activation. For activation by ImmHP or compound 1 , 30 μM Pf HGXPRT was preincubated with 30 μM ImmHP or compound 1 , 1.4 mM MgCl 2 , 1.4 mM pyrophosphate, and 50 mM potassium phosphate pH 7.4 in a 50 μL reaction mix for 30 min at 25 °C.…”