Calf spleen purine nucleoside phosphorylase: purification, sequence and crystal structure of its complex with an <i>N</i>(7)‐acycloguanosine inhibitor

Bzowska, Agnieszka; Luić, Marija; Schröder, Werner; Shugar, David; Saenger, Wolfram; Koellner, G.

doi:10.1016/0014-5793(95)00540-p

Cited by 53 publications

(48 citation statements)

References 33 publications

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“…I ) , which are known to be required for substrate binding [ l , 6, 341. The sequences and crystallographic structures of the binding sites of the calf spleen [37] and human erythrocyte [38] enzymes display close similarity. The crystallographic structure of a complex of the latter enzyme with ribavirin, a triazole-3-carboxamide riboside competitive inhibitor, has been resolved, and Fig.…”

Section: Discussionmentioning

confidence: 99%

Nicotinamide Riboside, an Unusual, Non‐Typical, Substrate of Purified Purine‐Nucleoside Phosphorylases

Wielgus‐Kutrowska

Kulikowska

Wierzchowski

et al. 1997

European Journal of Biochemistry

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Nicotinamide 1 -1l-D-riboside (Nir), the cationic, reducible moiety of the coenzyme NAD', has been confirmed as an unusual substrate for purified purine-nucleoside phosphorylase (PNP) from a mammalian source (calf spleen). It is also a substrate of the enzyme from Escherichia coli. The K,, values at pH 7, 1.48 mM and 0.62 mM, respectively, were 1-2 orders of magnitude higher than for the natural substrate inosine, but the V,,,,>, values were comparable, 96% and 35% that for Ino. The pseudo first-order rate constants, V,,,,,IK,,,, were 1 .I % and 2.5 o/o for the calf spleen and E. coli enzymes. The aglycon, nicotinamide, was neither a substrate nor an inhibitor of PNP.Nir was a weak inhibitor of inosine phosphorolysis catalyzed by both enzymes, with K, values close to the K,,, for its phosphorolysis, consistent with simple competitive inhibition; this was further confirmed by Dixon plots. Phosphorolysis of the fluorescent positively charged substrate 7-methylguanosine was also inhibited in a competitive manner by both Ino and Nir. Phosphorolysis of Nir by both enzymes was inhibited competitively by several specific inhibitors of calf spleen and E. coli PNP, with K, values similar to those for inhibition of other natural substrates.The pH dependence of the kinetic constants for the phosphorolysis of Nir and of a variety of other substrates, was extensively investigated, particularly in the alkaline pH range, where Nir exhibited abnormally high substrate activity relative to the reduced reaction rates of both enzymes towards other anionic or neutral substrates.The overall results are discussed in relation to present concepts regarding binding and phosphorolysis of substrates by PNP based on crystallographic data of enzyme-inhibitor complexes, and current studies on enzymatic and nonenzymatic mechanisms of the cleavage of the Nir glycosidic bond.Keywords; nicotinamide riboside ; purine-nucleoside phosphorylase; substratehnhibitor properties ; enzyme kinetics: pH dependence.The ubiquitous enzyme purine-nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine nucleosides such as guanosine and inosine and their deoxy counterparts in mammals [I], and additionally adenosine in some lower organisms such as Escherichiu coli and Salmonella typhimurium 121, as follows:Considerable attention is currently devoted to elucidation of the reaction mechanism, and development of potent specific inhibitors of the enzymes from various sources 13-51. Potential clinical applications of inhibitors involve treatment of T-cell leukemias, suppression of the host-versus-graft response in organ transplantations, and potentiation of the chemotherapeutic activity of PNP-cleavable nucleoside analogues 13, 61.Some non-natural purine-nucleoside analogues have been shown to be substrates, e.g. 7-alkyl congeners of guanosine and inosine [7], 7-P-D-and 3-P-D-purine ribosides [8, 91. while 8-azapurine analogues [lo], as well as the mesoionic anti-influenza agent 1,3,4-thiadiazol-2-ylcyanamide [ 11 1 undergo irreversible ribosy...

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Section: Discussionmentioning

confidence: 99%

Nicotinamide Riboside, an Unusual, Non‐Typical, Substrate of Purified Purine‐Nucleoside Phosphorylases

Wielgus‐Kutrowska

Kulikowska

Wierzchowski

et al. 1997

European Journal of Biochemistry

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“…acyclic substituted purines were described in a paper by Bzowska et al 16 The acyclic chain of the analogue 7-[(1,3-hydroxypropyl-2)amino]ethyl guanine is located at the 'non-natural' N 7 position of guanine and inhibits the enzyme purine nucleoside phosphorylase (PNP; purine nucleoside orthophosphate ribosyl transferase).…”

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confidence: 99%

Metastable and Collision-activated Decomposition of Protonated Molecules of Isomeric N7- and N9-Substituted Purines in the Gas Phase

Kralj

Žigon

Kocjan³

et al. 1997

Rapid Commun. Mass Spectrom.

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The structures and reactivities of protonated metastable and collision-activated molecules of isomeric N 7 -and N 9 -substituted purines were investigated using fast atom/ion bombardment ionization and mass analysed ion kinetic energy spectrometry methods. Calculated proton affinities (PAs) of isomeric purine derivatives and the intensities of the observed metastable-ion peaks revealed a direct correlation of PA with the reactivity of protonated molecules. The reactivity of the N 9 -substituted purines possessing N 3 -site specific proton affinities, i.e. N 3 -H + , relates to the strength of hydrogen bonding of N 3 -H + to oxygen atom(s) of the N 9 -substituent, e.g. CH 2 -O -CH 2 CH 2 -O -C(CH 3 ) = O or CH 2 -OCH 2 CH 2 -O -H. Various intramolecular ('intra-ion') hydrogen-bond(s) in the protonated molecules of isomeric purine derivates were revealed using proton affinities and metastable-ion peak intensities.

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“…A pnp1 null mutant (purchased from Euroscarf) did not show any obvious growth defect. We reasoned that if PNP activity is important for purine metabolism, the pnp1 mutant (18) and Bos taurus (2). Identities and similarities are highlighted in black and gray boxes, respectively.…”

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confidence: 99%

YLR209c Encodes Saccharomyces cerevisiae Purine Nucleoside Phosphorylase

et al. 2001

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The yeast YLR209c (PNP1) gene encodes a protein highly similar to purine nucleoside phosphorylases. This protein specifically metabolized inosine and guanosine. Disruption of PNP1 led to inosine and guanosine excretion in the medium, thus showing that PNP1 plays an important role in the metabolism of these purine nucleosides in vivo.Purine salvage is a complex pathway allowing interconversion of bases, nucleosides, and nucleotides. In yeast, major attention has been paid to the conversion of bases into nucleotides by phosphoribosyltransferases (PRTs): adenine-PRT, hypoxanthine-guanine-PRT, and xanthine-PRT activities have been reported (19,20), and the cognate genes have been identified (1,5,6). Yeast purine nucleoside metabolism has received far less attention, and only very recently was the first yeast gene encoding a purine nucleoside metabolizing enzyme identified (11). This gene, named ADO1, encodes adenosine kinase allowing synthesis of AMP from adenosine. Although several other enzymatic activities involved in yeast purine nucleoside metabolism have been described in the past, the corresponding genes have not yet been identified. Enzymatic activities responsible for the synthesis of inosine either from adenosine by adenosine deaminase (14) or from IMP by an IMP-specific 5Ј nucleotidase have been reported (8). Also, two distinct enzymatic activities (purine nucleoside hydrolase and purine nucleoside phosphorylase [PNP]) responsible for the degradation of inosine into hypoxanthine have been reported (7). The latter two enzymes catalyze the conversion of nucleosides to bases, although through distinct enzymatic mechanisms: (i) for nucleoside hydrolase, nucleoside ϩ H 2 O3base ϩ ribose and (ii) for nucleoside phosphorylase, nucleoside ϩ P i 3base ϩ ribose-1P.As a further step toward understanding yeast purine nucleoside metabolism, we searched for open reading frames (ORFs) in the complete yeast genome sequence that would encode candidate PNP. We found an uncharacterized ORF (YLR209c) that encodes a putative polypeptide highly similar to human and bovine PNP (Fig. 1). This enzyme has been thoroughly studied, and the three-dimensional structures of the trimeric human and bovine PNPs have been solved (3, 10). Important residues for substrate binding and catalysis have been identified (4, 12), all of which (except Val263) are conserved in the yeast enzyme (shown by asterisks in Fig. 1).To gain insight into the precise function of the yeast ORF YLR209c, the protein encoded by this ORF was tagged with 10 histidine residues at its N terminus and expressed in Escherichia coli. The PNP expression plasmid was constructed as follows. The YLR209c ORF was amplified by PCR from S288c genomic DNA with the following synthetic oligonucleotides: 359 (5Ј-CGATGCTCGAGATGAGTGATATCTTGAACGT-3Ј) and 360 (5Ј-GGACCCGGGTTATAATTCCCCCATTAC GG-3Ј). The amplification product was then digested with XhoI and SmaI and inserted in the pJC20-HisN vector (15) digested with XhoI and SmaI. The BL21(DE3) E. coli strain carrying the resulting plasmi...

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Calf spleen purine nucleoside phosphorylase: purification, sequence and crystal structure of its complex with an N(7)‐acycloguanosine inhibitor

Cited by 53 publications

References 33 publications

Nicotinamide Riboside, an Unusual, Non‐Typical, Substrate of Purified Purine‐Nucleoside Phosphorylases

Nicotinamide Riboside, an Unusual, Non‐Typical, Substrate of Purified Purine‐Nucleoside Phosphorylases

Metastable and Collision-activated Decomposition of Protonated Molecules of Isomeric N7- and N9-Substituted Purines in the Gas Phase

YLR209c Encodes Saccharomyces cerevisiae Purine Nucleoside Phosphorylase

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