Purine Nucleoside Phosphorylase

Stoeckler, Johanna D.; Parks, Robert E.

doi:10.1007/978-1-4684-4886-3_8

Cited by 20 publications

(4 citation statements)

References 28 publications

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“…The IC50 values were calculated from plots of % inhibition vs drug concentration (TableI). K-, values were determined from Lineweaver-Burk plots using the coupled xanthine oxidase assay 66. Values obtained are shown in TableIII.…”

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confidence: 99%

Structure-based design of inhibitors of purine nucleoside phosphorylase. 1. 9-(Arylmethyl) derivatives of 9-deazaguanine

Montgomery¹,

Niwas²,

Rose³

et al. 1993

J. Med. Chem.

130

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Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is a salvage enzyme important to the T-cell-mediated part of the immune system and as such is an important therapeutic target. This paper describes the design, synthesis, and enzymatic evaluation of potent, competitive inhibitors of PNP. Potential inhibitors were designed using the three-dimensional structure of the enzyme in an iterative process that involved interactive computer graphics to model the native enzyme and complexes of it with the inhibitors, Monte Carlo-based conformational searching, and energy minimization. Studies of the enzyme/inhibitor complexes were used to determine priorities of the synthetic efforts. The resulting compounds were then evaluated by determination of their IC50 values and by X-ray diffraction analysis using difference Fourier maps. In this manner, we have developed a series of 9-(arylmethyl)-9-deazapurines (2-amino-7-(arylmethyl)-4H-pyrrolo[3,2-d]-pyrimidin-4-ones) that are potent, membrane-permeable inhibitors of the enzyme. The IC50 values of these compounds range from 17 to 270 nM (in 1 mM phosphate), with 9-(3,4-dichlorobenzyl)-9-deazaguanine being the most potent inhibitor. X-ray analysis explained the role of the aryl groups and revealed the rearrangement of hydrogen bonds in the binding of the 9-deazaguanines in the active site of PNP relative to the binding of the 8-aminoguanines that results in more potent inhibition of the enzyme.

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confidence: 99%

Structure-based design of inhibitors of purine nucleoside phosphorylase. 1. 9-(Arylmethyl) derivatives of 9-deazaguanine

Montgomery¹,

Niwas²,

Rose³

et al. 1993

J. Med. Chem.

130

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“…Up to now, many efforts have been made for the assay of PNP activity (Adam et al, 1998;Kalckar, 1947;Stoeckler and Parks, 1985;Wierzchowski et al, 1996Wierzchowski et al, , 2002. The most widely used method proposed by Kalckar is based on the couple of xanthine oxidase, which oxidizes the hypoxanthine released by phosphorolysis of inosine to uric acid.…”

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confidence: 99%

Sensing purine nucleoside phosphorylase activity by using silver nanoparticles

Cao

Wang

et al. 2010

Biosensors and Bioelectronics

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“…PNP‐1 are found in prokaryotes, are homohexamers with a subunit of ∼26 kDa and recognize both 6‐oxo and 6‐amino purine nucleosides as substrates. PNP‐2 are homotrimers, with a subunit molecular mass of ∼30 kDa and accept only guanosine and inosine as substrates [3–5]. It is interesting to note that many organisms that express PNP‐1 also express PNP‐2 [5].…”

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confidence: 99%

“…Abbreviations hMTAP, human 5¢-deoxy-5¢-methylthioadenosine phosphorylase; MTA, 5¢-deoxy-5¢-methylthioadenosine; MTAP, 5¢-deoxy-5¢methylthioadenosine phosphorylase; PfMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Pyrococcus furiosus; PfPNP, purine nucleoside phosphorylase from P. furiosus; PNP, purine nucleoside phosphorylase; SsMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Sulfolobus solfataricus; SsMTAPII, 5¢-deoxy-5¢-methylthioadenosine phosphorylase II from S. solfataricus. 30 kDa and accept only guanosine and inosine as substrates [3][4][5]. It is interesting to note that many organisms that express PNP-1 also express PNP-2 [5].…”

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confidence: 99%

Biochemical and structural characterization of mammalian‐like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus

Cacciapuoti¹,

Gorassini²,

Mazzeo

et al. 2007

The FEBS Journal

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Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolytic cleavage of the glycosidic bond of purine nucleosides to produce ribose-1-phosphate and a free purine base [1][2][3]. PNPs have been characterized in a variety of species and may be grouped into two main groups, PNP-1 and PNP-2. PNP-1 are found in prokaryotes, are homohexamers with a subunit of 26 kDa and recognize both 6-oxo and 6-amino purine nucleosides as substrates. PNP-2 are homotrimers, with a subunit molecular mass of We report here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus (PfPNP). The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. PfPNP is a homohexamer of 180 kDa which shows a much higher similarity with 5¢-deoxy-5¢-methylthioadenosine phosphorylase (MTAP) than with purine nucleoside phosphorylase (PNP) family members. Like human PNP, PfPNP shows an absolute specificity for inosine and guanosine. PfPNP shares 50% identity with MTAP from P. furiosus (PfMTAP). The alignment of the protein sequences of PfPNP and PfM-TAP indicates that only four residue changes are able to switch the specificity of PfPNP from a 6-oxo to a 6-amino purine nucleoside phosphorylase still maintaining the same overall active site organization. PfPNP is highly thermophilic with an optimum temperature of 120°C and is characterized by extreme thermodynamic stability (T m , 110°C that increases to 120°C in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100°C), and remarkable SDS-resistance. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. By integrating biochemical methodologies with mass spectrometry we assigned three pairs of intrasubunit disulfide bridges that play a role in the stability of the enzyme against thermal inactivation. The characterization of the thermal properties of the C254S ⁄ C256S mutant suggests that the CXC motif in the C-terminal region may also account for the extreme enzyme thermostability.Abbreviations hMTAP, human 5¢-deoxy-5¢-methylthioadenosine phosphorylase; MTA, 5¢-deoxy-5¢-methylthioadenosine; MTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase; PfMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Pyrococcus furiosus; PfPNP, purine nucleoside phosphorylase from P. furiosus; PNP, purine nucleoside phosphorylase; SsMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Sulfolobus solfataricus; SsMTAPII, 5¢-deoxy-5¢-methylthioadenosine phosphorylase II from S. solfataricus.

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Purine Nucleoside Phosphorylase

Cited by 20 publications

References 28 publications

Structure-based design of inhibitors of purine nucleoside phosphorylase. 1. 9-(Arylmethyl) derivatives of 9-deazaguanine

Structure-based design of inhibitors of purine nucleoside phosphorylase. 1. 9-(Arylmethyl) derivatives of 9-deazaguanine

Sensing purine nucleoside phosphorylase activity by using silver nanoparticles

Biochemical and structural characterization of mammalian‐like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus

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