Purine nucleoside phosphorylase activity decline is linked to the decay of the trimeric form of the enzyme

Wielgus‐Kutrowska, Beata; Modrak-Wójcik, Anna; Dyzma, Alicja; Breer, Katarzyna; Żółkiewski, Michał; Bzowska, Agnieszka

doi:10.1016/j.abb.2014.03.009

Cited by 7 publications

(8 citation statements)

References 44 publications

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“…This molecular weight was reported in literature for the enzyme monomer (Wielgus‐Kutrowska et al . ) and also in the data sheet of the recombinant purified human protein, used as internal standard that, as expected, showed an identical migration pattern (Fig. a1).…”

Section: Resultssupporting

confidence: 80%

“…In an attempt to shed light on these aspects, we initially focused on purine nucleoside phosphorylase (PNP, EC 2.4.2.1) that is commonly considered a cytoplasmic enzyme in prokaryotic and eukaryotic organisms (Wielgus‐Kutrowska et al . ). A localization in mitochondria of bovine liver (Haag and Lewis ) has also been reported, although for its several unique properties mitochondrial PNP is considered to be a different protein from the cytosolic one.…”

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confidence: 97%

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Evidence for purine nucleoside phosphorylase (PNP) release from rat C6 glioma cells

Giuliani

Zuccarini

Buccella

et al. 2017

Journal of Neurochemistry

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Intracellular purine turnover is mainly oriented to preserving the level of triphosphate nucleotides, fundamental molecules in vital cell functions that, when released outside cells, act as receptor signals. Conversely, high levels of purine bases and uric acid are found in the extracellular milieu, even in resting conditions. These compounds could derive from nucleosides/bases that, having escaped to cell reuptake, are metabolized by extracellular enzymes similar to the cytosolic ones. Focusing on purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of purine (deoxy)-nucleosides/bases, we found that it is constitutively released from cultured rat C6 glioma cells into the medium, and has a molecular weight and enzyme activity similar to the cytosolic enzyme. Cell exposure to 10 μM ATP or guanosine triphosphate (GTP) increased the extracellular amount of all corresponding purines without modifying the levels/activity of released PNP, whereas selective activation of ATP P2Y or adenosine A metabotropic receptors increased PNP release and purine base formation. The reduction to 1% in oxygen supply (2 h) to cells decreased the levels of released PNP, leading to an increased presence of extracellular nucleosides and to a reduced formation of xanthine and uric acid. Conversely, 2 h cell re-oxygenation enhanced the extracellular amounts of both PNP and purine bases. Thus, hypoxia and re-oxygenation modulated in opposite manner the PNP release/activity and, thereby, the extracellular formation of purine metabolism end-products. In conclusion, extracellular PNP and likely other enzymes deputed to purine base metabolism are released from cells, contributing to the purinergic system homeostasis and exhibiting an important pathophysiological role.

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Section: Resultssupporting

confidence: 80%

mentioning

confidence: 97%

Evidence for purine nucleoside phosphorylase (PNP) release from rat C6 glioma cells

Giuliani

Zuccarini

Buccella

et al. 2017

Journal of Neurochemistry

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“…It can be noted that the possibility that the enzyme also displays activity in the monomeric form exists . However, a recent study shows that PNP monomers are unstable in solution, and consequently, packing of monomers into trimers is necessary for enzyme stability . The phosphate loop was here modeled in the closed conformation, resulting in His64 being organized in the active site within interacting distance of Ser33 and His86.…”

Section: Resultsmentioning

confidence: 99%

“…54 However, a recent study shows that PNP monomers are unstable in solution, and consequently, packing of monomers into trimers is necessary for enzyme stability. 55 The phosphate loop was here modeled in the closed conformation, resulting in His64 being organized in the active site within interacting distance of Ser33 and His86. All EVB simulations were repeated 100 times, resulting in a total simulation time of 51 ns for each substrate.…”

Section: H 2 Pomentioning

confidence: 99%

Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase

et al. 2016

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Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity.

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“…(a) (b) In our recent studies of the trimeric calf PNP natural aging process, we have show that the loss of enzymatic activity of the wild-type enzyme correlates with the degradati of its trimeric structure, subsequently leading to aggregation. However, we have not o served individual, stable subunits as intermediates of the obsolescence process [20].…”

Section: Introductionmentioning

confidence: 99%

Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme

Dyzma

Wielgus‐Kutrowska

Girstun

et al. 2023

IJMS

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Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the enzyme. Molecular dynamics simulations showed different geometries of the active site in the non-mutated trimeric and monomeric PNP forms, which suggested that the active site in the isolated monomer could be non-functional. To confirm this hypothesis, six amino acids located at the interface of the subunits were selected and mutated to alanines to disrupt the trimer and obtain a monomer (6Ala PNP). The effects of these mutations on the enzyme structure, stability, conformational dynamics, and activity were examined. The solution experiments confirmed that the 6Ala PNP mutant occurs mainly as a monomer, with a secondary structure almost identical to the wild type, WT PNP, and importantly, it shows no enzymatic activity. Simulations confirmed that, although the secondary structure of the 6Ala monomer is similar to the WT PNP, the positions of the amino acids building the 6Ala PNP active site significantly differ. These data suggest that a trimeric structure is necessary to stabilize the geometry of the active site of this enzyme.

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Purine nucleoside phosphorylase activity decline is linked to the decay of the trimeric form of the enzyme

Cited by 7 publications

References 44 publications

Evidence for purine nucleoside phosphorylase (PNP) release from rat C6 glioma cells

Evidence for purine nucleoside phosphorylase (PNP) release from rat C6 glioma cells

Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase

Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme

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