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

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Abstract

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 phosphoribosyltransferase (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 DNAN, SN1-like transition state.

Original languageEnglish (US)
Pages (from-to)6368-6376
Number of pages9
JournalBiochemistry
Volume56
Issue number48
DOIs
StatePublished - Dec 5 2017

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Plasmodium falciparum
Isotopes
Hypoxanthine
Kinetics
Purinones
Parasites
Nucleotides
Purine Nucleotides
Salvaging
Purines
Charge distribution
Bond length
Metabolism
Malaria
Labels
Atoms
Geometry
hypoxanthine-guanine-xanthine phosphoribosyltransferase
diphosphoric acid
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Plasmodium falciparum",
abstract = "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 phosphoribosyltransferase (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 DNAN, SN1-like transition state.",
author = "Ducati, {Rodrigo G.} and Firestone, {Ross S.} and Schramm, {Vern L.}",
year = "2017",
month = "12",
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doi = "10.1021/acs.biochem.7b01027",
language = "English (US)",
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publisher = "American Chemical Society",
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TY - JOUR

T1 - Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Plasmodium falciparum

AU - Ducati, Rodrigo G.

AU - Firestone, Ross S.

AU - Schramm, Vern L.

PY - 2017/12/5

Y1 - 2017/12/5

N2 - 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 phosphoribosyltransferase (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 DNAN, SN1-like transition state.

AB - 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 phosphoribosyltransferase (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 DNAN, SN1-like transition state.

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