Human hypoxanthine-guanine phosphoribosyltransferase. Steady state kinetics of the forward and reverse reactions.

Giacomello, Alessandro; Salerno, C

doi:10.1016/s0021-9258(17)34576-3

Cited by 79 publications

(36 citation statements)

References 23 publications

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“…1), the residues of which play important roles in the processes of substrate binding, catalysis and product release. [8][9][10][11][12][13] The kinetic mechanism of human 14,15 and Schistosoma mansoni 16 HGPRTases revealed ordered substrate binding wherein the binding of PRPP precedes the binding of hypoxanthine/guanine. The release of products is also sequential and involves the leaving of PPi prior to IMP/GMP/XMP.…”

Section: Introductionmentioning

confidence: 99%

“…The release of products is also sequential and involves the leaving of PPi prior to IMP/GMP/XMP. [12][13][14][15][16] The processes of substrate binding, catalysis and product release involve a large number of conformational changes in the active site loops, which have been investigated in the cases of Toxoplasma gondii HGXPRT 12 and human HGPRT. 13 These changes are largely similar in the two enzymes, with the first event being the isomerization of the Leu78-Lys79 (T. gondii numbering) peptide bond (in loop I), which creates a cavity for PRPP binding.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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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.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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“…Several experiments have been carried out to investigate substrate binding and product release in HsHGPRT. , However, a detailed molecular-level description and its dynamical characteristics have not been explored until now. Specifically, the sequence of release of IMP and PPi.2Mg has been investigated experimentally, and it has been suggested that the latter is released first .…”

Section: Discussionmentioning

confidence: 99%

“…The order of release of the products (IMP and PPi.2Mg) is somewhat unclear. Giacomello and Salerno proposed a random release, while Xu et al proposed sequential substrate binding and partially random dissociation of the products. The kinetic mechanism of PfHGXPRT has recently been examined thoroughly; the same was investigated earlier for homologous enzymes from other species. , The rate of dissociation of IMP ( k –3 ) from a double mutant (W181S/F197W) of PfHGXPRT was determined to be 33 s –1 , which corresponds to a release time approximately of 30 ms .…”

Section: Introductionmentioning

confidence: 99%

“…26 The order of release of the products (IMP and PPi.2Mg) is somewhat unclear. Giacomello and Salerno 27 proposed a random release, while Xu et al 26 proposed sequential substrate binding and partially random dissociation of the products. The kinetic mechanism of PfHGXPRT has recently been examined thoroughly; 28 the same was investigated earlier for homologous enzymes from other species.…”

Section: ■ Introductionmentioning

confidence: 99%

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Product Release Pathways in Human and Plasmodium falciparum Phosphoribosyltransferase

Karmakar

Roy

Balaram

et al. 2016

J. Chem. Inf. Model.

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Atomistic molecular dynamics (MD) simulations coupled with the metadynamics technique were carried out to delineate the product (PPi.2Mg and IMP) release mechanisms from the active site of both human (Hs) and Plasmodium falciparum (Pf) hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT). An early movement of PPi.2Mg from its binding site has been observed. The swinging motion of the Asp side chain (D134/D145) in the binding pocket facilitates the detachment of IMP, which triggers the opening of flexible loop II, the gateway to the bulk solvent. In PfHGXPRT, PPi.2Mg and IMP are seen to be released via the same path in all of the biased MD simulations. In HsHGPRT too, the product molecules follow similar routes from the active site; however, an alternate but minor escape route for PPi.2Mg has been observed in the human enzyme. Tyr 104 and Phe 186 in HsHGPRT and Tyr 116 and Phe 197 in PfHGXPRT are the key residues that mediate the release of IMP, whereas the motion of PPi.2Mg away from the reaction center is guided by the negatively charged Asp and Glu and a few positively charged residues (Lys and Arg) that line the product release channels. Mutations of a few key residues present in loop II of Trypanosoma cruzi (Tc) HGPRT have been shown to reduce the catalytic efficiency of the enzyme. Herein, in silico mutation of corresponding residues in loop II of HsHGPRT and PfHGXPRT resulted in partial opening of the flexible loop (loop II), thus exposing the active site to bulk water, which offers a rationale for the reduced catalytic activity of these two mutant enzymes. Investigations of the product release from these HsHGPRT and PfHGXPRT mutants delineate the role of these important residues in the enzymatic turnover.

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Guanine nucleotide depletion triggers cell cycle arrest and apoptosis in human neuroblastoma cell lines

Messina

Gazzaniga

Micheli

et al. 2004

Intl Journal of Cancer

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Mycophenolic acid (MPA) specifically inhibits inosine-5 -monophosphate dehydrogenase, the first committed step toward GMP biosynthesis. In its morpholinoethyl ester prodrug form it is one of the most promising immunosuppressive drugs recently developed. The aim of the present study was to investigate the in vitro effects of MPA, at concentrations readily attainable during immunosuppressive therapy, on 3 human neuroblastoma cell lines (LAN5, SHEP and IMR32). Mycophenolic acid (0.1-10 M) caused a decrease of intracellular levels of guanine nucleotides, a G 1 arrest and a time-and dose-dependent death by apoptosis. These effects, associated with an up-regulation of p53, p21 and bax, a shuttling of p53 protein into the nucleus and a down-regulation of bcl-2, survivin and p27 protein, were reversed by the simultaneous addition of guanine or guanosine and were more evident using nondialysed serum containing hypoxanthine. These results suggest that in neuroblastoma cell lines clinically attainable concentrations of mycophenolic acid deplete guanine nucleotide pools triggering G 1 arrest and apoptosis through p53-mediated pathways, indicating a potential role of its morpholinoethyl ester pro-drug in the management of patients with neuroectodermal tumors.

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Human hypoxanthine-guanine phosphoribosyltransferase. Steady state kinetics of the forward and reverse reactions.

Cited by 79 publications

References 23 publications

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

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

Product Release Pathways in Human and Plasmodium falciparum Phosphoribosyltransferase

Guanine nucleotide depletion triggers cell cycle arrest and apoptosis in human neuroblastoma cell lines

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