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Role of ionization of the phosphate cosubstrate on phosphorolysis by purine nucleoside phosphorylase (PNP) of bacterial (E. coli) and mammalian (human) origin

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Abstract

Kinetics of the reactions of purine nucleoside phosphorylases (PNP) from E. coli (PNP-I, the product of the deoD gene) and human erythrocytes with their natural substrates guanosine (Guo), inosine (Ino), a substrate analogue N(7)-methylguanosine (m7Guo), and orthophosphate (Pi, natural cosubstrate) and its thiophosphate analogue (SPi), found to be a weak cosubstrate, have been studied in the pH range 5–8. In this pH range Guo and Ino exist predominantly in the neutral forms (pKa 9.2 and 8.8); m7Guo consists of an equilibrium mixture of the cationic and zwitterionic forms (pKa 7.0); and Pi and SPi exhibit equilibria between monoanionic and dianionic forms (pKa 6.7 and 5.4, respectively). The phosphorolysis of m7Guo (at saturated concentration) with both enzymes exhibits Michaelis kinetics with SPi, independently of pH. With Pi, the human enzyme shows Michaelis kinetics only at pH ∼5. However, in the pH range 5–8 for the bacterial enzyme, and 6–8 for the human enzyme, enzyme kinetics with Pi are best described by a model with high- and low-affinity states of the enzymes, denoted as enzyme-substrate complexes with one or two active sites occupied by Pi, characterized by two sets of enzyme-substrate dissociation constants (apparent Michaelis constants, K m1 and K m2) and apparent maximal velocities (V max1 and V max2). Their values, obtained from non-linear least-squares fittings of the Adair equation, were typical for negative cooperativity of both substrate binding (K m1 < K m2) and enzyme kinetics (V max1/K m1 > V max2/K m2). Comparison of the pH-dependence of the substrate properties of Pi versus SPi points to both monoanionic and dianionic forms of Pi as substrates, with a marked preference for the dianionic species in the pH range 5–8, where the population of the Pi dianion varies from 2 to 95%, reflected by enzyme efficiency three orders of magnitude higher at pH 8 than that at pH 5. This is accompanied by an increase in negative cooperativity, characterized by a decrease in the Hill coefficient from n H ∼1 to n H ∼0.7 for Guo with the human enzyme, and to n H ∼0.7 and 0.5 for m7Guo with the E. coli and human enzymes, respectively. Possible mechanisms of cooperativity are proposed. Attention is drawn to the substrate properties of SPi in relation to its structure.

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Abbreviations

PNP:

Purine nucleoside phosphorylase

PNP-I and PNP-II:

PNPs from E. coli, the products of the deoD and xapA genes, respectively

m7Guo:

N(7)-methylguanosine

FA:

Formycin A

m6FA:

N(6)-methylformycin A

m7FA:

N(7)-methylformycin A

Hepes:

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

Mes:

2-[N-morpholino]ethanesulfonic acid

Pi :

Orthophosphate

SPi :

Thiophosphate

References

  • Ames BN (1966) Assay of inorganic phosphate. Methods Enzymol 8:115–116

    Google Scholar 

  • Behlke J, Koellner G, Bzowska A (2005) Oligomeric structure of mammalian purine nucleoside phosphorylase in solution determined by analytical ultracentrifugation. Z Naturforsch 60C:927–931

    Google Scholar 

  • Bennett EM, Li C, Allan PW, Parker WB, Ealick SE (2003) Structural basis for substrate specificity of Escherichia coli purine nucleoside phosphorylase. J Biol Chem 278:47110–47118

    Article  Google Scholar 

  • Bzowska A, Kazimierczuk Z, Seela F (1998) 7-Deazapurine 2′-deoxyribofuranosides are noncleavable competitive inhibitors of Escherichia coli purine nucleoside phosphorylase (PNP). Acta Biochim Polon 45:755–768

    Google Scholar 

  • Bzowska A, Kulikowska E, Shugar D (2000) Purine nucleoside phosphorylases: properties, functions, and clinical aspects. Pharmacol Ther 88:349–425

    Article  Google Scholar 

  • Bzowska A (2002) Calf spleen purine nucleoside phosphorylase: complex kinetic mechanism, hydrolysis of 7-methylguanosine, and oligomeric state in solution. Biochim Biophys Acta 1596:293–317

    Google Scholar 

  • Bzowska M, Stępniak K, Olasek M, Długosz M, Wielgus-Kutrowska B, Antosiewicz J, Holy A, Koellner G, Stroh A, Raszewski G, Steiner T, Frank J (2005) Attempts to differentiate subunits of trimeric and hexameric purine nucleoside phosphorylases by crystal structure and solution studies using purine bases, modified purine nucleosides, acyclonucleosides and their phosphonate analogues. Collection Czechoslov Chem Commun 7:133–142

    Google Scholar 

  • Canduri F, dos Santos DM, Silva RG, Mendes MA, Basso LA, Palma MS, de Axevedo WF, Santos DS (2004) Structures of human purine nucleoside phosphorylase complexed with inosine and ddI. Biochem Biophys Res Commun 313:907–914

    Article  Google Scholar 

  • Chlebowski JF, Coleman JE (1974) Mechanisms of hydrolysis of O-phosphorothioates and inorganic thiophosphate by Escherichia coli alkaline phosphatase. J Biol Chem 249:7192–7202

    Google Scholar 

  • Choi H-S, Stoeckler JD, Parks RE Jr (1986) 5-Iodoribose 1-phosphate, an analog of ribose 1-phosphate. Enzymatic synthesis and kinetic studies with enzymes of purine, pyrimidine, and sugar phosphate metabolism. J Biol Chem 261:599–607

    Google Scholar 

  • Dawson RMC, Elliott DC, Elliott WH, Jones KM (eds) (1969) Spectral data and pK values for purines, pyrimidines, nucleosides and nucleotides. In: Data for biochemical research. Oxford University Press, Oxford, p 176

  • Dandanell G, Szczepanowski R, Kierdaszuk B, Shugar D, Bochtler M (2005) Escherichia coli purine nucleoside phosphorylase II, the product of the xapA gene. J Mol Biol 348:113–125

    Article  Google Scholar 

  • de Azevedo WF Jr, Canduri F, Dos Santos DM, Pereira JH, Dias MVB, Silva RG, Mendes MA, Palma MS, Basso LA, Santos DS (2003) Structural basis for inhibition of human PNP by immucillin-H. Biochem Biophys Res Commun 309:917–922

    Article  Google Scholar 

  • Deng H, Lewandowicz A, Schramm VL, Callender R (2004) Activating the phosphate nucleophile at the catalytic site of purine nucleoside phosphorylase: a vibrational spectroscopic study. J Am Chem Soc 126:9516–9517

    Article  Google Scholar 

  • Deng H, Murkin AS, Schramm VL (2006) Phosphate activation in the ground state of purine nucleoside phosphorylase. J Am Chem Soc 128:7765–7771

    Article  Google Scholar 

  • Doskocil J, Holy A (1977) Specificity of purine nucleoside phosphorylase from Escherichia coli. Collection Czechoslov Chem Commun 42:370–383

    Google Scholar 

  • Ealick SE, Rule SA, Carter DC, Greenhough TJ, Babu YS, Cook WJ, Habash J, Helliwell JR, Stoeckler JD, Parks RE Jr, Chen S, Bugg CE (1990) Three-dimensional structure of human erythrocytic purine nucleoside phosphorylase at 3.2 Å resolution. J Biol Chem 265:1812–1820

    Google Scholar 

  • Erion MD, Takabayashi K, Smith HB, Kessi J, Wagner S, Hönger S, Shames SL, Ealick SE (1997a) Purine nucleoside phosphorylase. 1. Structure-function studies. Biochemistry 36:11725–34

    Article  Google Scholar 

  • Erion MD, Stoeckler JD, Guida WC, Walter L, Ealick SE (1997b) Purine nucleoside phosphorylase. 2. Catalytic mechanism. Biochemistry 36:11735–11748

    Article  Google Scholar 

  • Frey PA, Sammons RD (1985) Bond order and charge localization in nucleoside phosphorothioates. Science 228:541–545

    Article  ADS  Google Scholar 

  • Frey PA (1989) Chiral phosphorothioates: stereochemical analysis of enzymatic substitution at phosphorus. Adv Enzymol Relat Areas Mol Biol 62:119–201

    Article  Google Scholar 

  • Gerlt JA, Demou PC, Mehdi S (1982) Oxygen-17 NMR spectral properties of simple phosphate esters and adenine nucleotides. J Am Chem Soc 104:2848–2856

    Article  Google Scholar 

  • Golos B, Dzik JM, Kazimierczuk Z, Ciesla J, Zielinski Z, Jankowska J, Kraszewski A, Stawinski J, Rode W, Shugar D (2001) Interaction of thymidylate synthase with the 5′-thiophosphates, 5′-dithiophosphates, 5′-H-phosphonates and 5′-S-thiosulfates of 2′-deoxyuridine, thymidine and 5-fluoro-2′-deoxyuridine. Biol Chem 382:1439–1445

    Article  Google Scholar 

  • Hendler SS, Fuerer E, Srinivasan PR (1970) Synthesis and chemical properties of monomers and polymers containing 7-methylguanine and an investigation of their substrate or template properties for bacterial deoxyribonucleic acid or ribonucleic acid polymerases. Biochemistry 9:4141–4153

    Article  Google Scholar 

  • Jaffe EK, Cohn M (1978) 31P nuclear magnetic resonance spectra of the thiophosphate analogs of adenine nucleotides; effects of pH and Mg2+ binding. Biochemistry 17:652–657

    Article  Google Scholar 

  • Jensen KF, Nygaard P (1975) Purine nucleoside phosphorylase from Escherichia coli and Salmonella typhimurium. Purification and some properties. Eur J Biochem 51:253–265

    Article  Google Scholar 

  • Jensen KF (1976) Purine nucleoside phosphorylase from Escherichia coli and Salmonella typhimurium. Initial velocity kinetics, ligand binding and reaction mechanism. Eur J Biochem 61:377–386

    Article  Google Scholar 

  • Jungas RL (2006) Best literature values for the pK of carbonic and phosphoric acid under physiological conditions. Anal Biochem 349:1–15

    Article  Google Scholar 

  • Kicska GA, Tyler PC, Evans GB, Furneaux RH, Shi W, Fedorov A, Lewandowicz A, Cahill SM, Almo SC, Schramm VL (2002) Atomic dissection of the hydrogen bond network for transition-state analogue binding to purine nucleoside phosphorylase. Biochemistry 41:14489–14498

    Article  Google Scholar 

  • Kierdaszuk B, Modrak-Wójcik A, Shugar D (1997) Binding of phosphate and sulfate anions by purine nucleoside phosphorylase from E coli: ligand dependent quenching of enzyme intrinsic fluorescence. Biophys Chem 63:107–118

    Article  Google Scholar 

  • Kierdaszuk B, Modrak-Wójcik A, Wierzchowski J, Shugar D, (2000) Formycin A and its N-methyl analogues, specific inhibitor of E coli purine nucleoside phosphorylase (PNP): induced tautomeric shifts on binding to enzyme, and enzyme-ligand fluorescence resonance energy transfer. Biochim Biophys Acta 1476:109–128

    Google Scholar 

  • Kline PC, Schramm VL (1992) Purine nucleoside phosphorylase. Inosine hydrolysis, tight binding of the hypoxanthine intermediate, and third-the-sites reactivity. Biochemistry 31:5964–5973

    Article  Google Scholar 

  • Kline PC, Schramm VL (1995) Pre-steady-state transition-state analysis of the hydrolytic reaction catalyzed by purine nucleoside phosphorylase. Biochemistry 34:1153–1162

    Article  Google Scholar 

  • Koellner G, Luić M, Shugar D, Seanger W, Bzowska A (1997) Crystal structure of calf spleen purine nucleoside phosphorylase in a complex with hypoxanthine at 2.15 Å resolution. J Mol Biol 265:202–216

    Article  Google Scholar 

  • Koellner G, Luić M, Shugar D, Seanger W, Bzowska A (1998) Crystal structure of the ternary complex of E coli purine nucleoside phosphorylase with formycin B, a structural analogue of the substrate inosine, and phosphate (sulphate) at 2.1 Å resolution. J Mol Biol 280:153–166

    Article  Google Scholar 

  • Koellner G, Bzowska A, Wielgus-Kutrowska B, Luić M, Steiner T, Seanger W, Stępiński J (2002) Open and closed conformation of the E coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism. J Mol Biol 315:351–371

    Article  Google Scholar 

  • Koshland DE, Némethy G, Filmer D (1966) Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry 5:365–385

    Article  Google Scholar 

  • Krenitsky TA, Tuttle JV, Miller WH, Moorman AR, Orr GF, Beauchamp L (1990) Nucleotide analogue inhibitors of purine nucleoside phosphorylase. J Biol Chem 265:3066–3069

    Google Scholar 

  • Kulikowska E, Bzowska A, Holy A, Magnowska L, Shugar D (1998) Antiviral acyclic nucleoside phosphonate analogues as inhibitors of purine nucleoside phosphorylases. Adv Exp Med Biol 431:747–752

    Google Scholar 

  • Lewandowicz A, Schramm VL (2004) Transition state analysis for human and Plasmodium falciparum purine nucleoside phosphorylases. Biochemistry 43:1458–1468

    Article  Google Scholar 

  • Mao C, Cook WJ, Zhou M, Koszalka GW, Krenitsky TA, Ealick SE (1997) The crystal structure of E coli purine nucleoside phosphorylase: a comparison with human enzyme reveals a conserved topology. Structure 5:1373–1383

    Article  Google Scholar 

  • Mao C, Cook WJ, Zhou M, Federov AA, Almo SC, Ealick SE (1998) Calf spleen purine nucleoside phosphorylase complexed with substrates and substrate analogues. Biochemistry 37:7135–7146

    Article  Google Scholar 

  • Modrak-Wójcik A, Stępniak K, Akoev V, Żółkiewski M, Bzowska A (2006) Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy. Protein Sci 15:1794–1800

    Article  Google Scholar 

  • Naito Y, Lowenstein JM (1985) 5′-Nucleotidase from rat heart membranes. Inhibition by adenine nucleotides and related compounds. Biochem J 226:645–651

    Google Scholar 

  • Narayana SVL, Bugg CE, Ealick SE (1997) Refined structure of purine nucleoside phosphorylase at 2.75 Å resolution. Acta Crystallog D 53:131–142

    Article  Google Scholar 

  • Neet KE (1980) Cooperativity in enzyme function: equilibrium and kinetic aspects. Methods Enzymol 64:139–192

    Google Scholar 

  • Pugmire MJ, Ealick SE (2002) Structural analyses reveal two distinct families of nucleoside phosphorylases. Biochem J 361:1–25

    Article  Google Scholar 

  • Ropp PA, Traut TW (1991) Purine nucleoside phosphorylase. Allosteric regulation of a dissociating enzyme. J Biol Chem 266:7682–7687

    Google Scholar 

  • Sauve AA, Cahill SM, Zech SG, Basso LA, Lewandowicz A, Santos DS, Grubmeyer Ch, Evans GB, Furneaux RH, Tyler PC, McDermott A, Girvin ME, Schramm VL (2003) Ionic states of substrates and transition state analogues at the catalytic sites of N-ribosyltransferases. Biochemistry 42:5694–5705

    Article  Google Scholar 

  • Schnick C, Robien MA, Brzozowski AM, Dodson EJ, Murshudov GN, Anderson L, Luft JR, Mehlin C, Hol WG, Brannigan JA, Wilkinson AJ (2005) Structures of Plasmodium falciparum purine nucleoside phosphorylase complexed with sulfate and its natural substrate inosine. Acta Crystallogr D Biol Crystallogr 61:1245–1254

    Article  Google Scholar 

  • Stoeckler JD, Agarwal RP, Agarwal KC, Schmid K, Parks RE Jr (1978a) Purine nucleoside phosphorylase from human erythrocytes: physicochemical properties of the crystalline enzyme. Biochemistry 17:278–283

    Article  Google Scholar 

  • Stoeckler JD, Agarwal RP, Agarwal KC, Parks RE Jr (1978b) Purine nucleoside phosphorylase from human erythrocytes. Methods Enzymol 51:530–538

    Article  Google Scholar 

  • Stoychev G, Kierdaszuk B, Shugar D (2001) Interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with the cationic and zwitterionic forms of the fluorescent substrate N(7)-methylguanosine. Biochim Biophys Acta 1544:74–88

    Google Scholar 

  • Stoychev G, Kierdaszuk B, Shugar D (2002) Xanthosine and xanthine. Substrate properties with purine nucleoside phosphorylases, and relevance to other enzyme systems. Eur J Biochem 269:4048–4057

    Article  Google Scholar 

  • Taylor Ringia EA, Schramm VL (2006) Transition states and inhibitors of the purine nucleoside phosphorylase family. Curr Top Med Chem 5:1237–1258

    Article  Google Scholar 

  • Traut TW (1994) Physiological concentration of purines and pyrimidines. Mol Cell Biochem 140:1–22

    Article  Google Scholar 

  • Tuttle JV, Krenitsky TA (1984) Effects of acyclovir and its metabolites on purine nucleoside phosphorylase. J Biol Chem 259:4065–4069

    Google Scholar 

  • Wielgus-Kutrowska B, Tebbe J, Schröder W, Luić M, Shugar D, Seanger W, Koellner G, Bzowska A (1998) Cellulomonas sp. purine nucleoside phosphorylase (PNP). Comparison with human and E. coli enzymes. Adv Exp Med Biol 431:259–264

    Google Scholar 

  • Wierzchowski J, Shugar D (1982) Luminescence studies of formycin, its aglycone, and their N-methyl derivatives: tautomerisation, sites of protonation and phototautomerism. Photochem Photobiol 35:445–458

    Article  Google Scholar 

  • Williams SR, Gekeler V, McIvor RS, Martin DW Jr (1987) A human purine nucleoside phosphorylase deficiency caused by a single base change. J Biol Chem 262:2332–2338

    Google Scholar 

  • Wlodarczyk J, Stoychev Galitonov G, Kierdaszuk B (2004) Identification of the tautomeric form of formycin A in its complex with E coli purine nucleoside phosphorylase based on the effect of enzyme-ligand binding on fluorescence and phosphorescence. Eur Biophys J 33:377–385

    Article  Google Scholar 

  • Zhao Z (1996) Thiophosphate derivatives as inhibitors of tyrosine phosphatases. Biochem Biophys Res Commun 218:480–484

    Article  Google Scholar 

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Acknowledgment

We are indebted to Prof. Ryszard Stolarski (Department of Biophysics, University of Warsaw) for critical reading of the manuscript. This investigation was supported by the Polish Ministry of Scientific Research and Higher Education (MNSzW), grant No. 3P04A02425.

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Authors and Affiliations

  1. Department of Biophysics, Institute of Experimental Physics, University of Warsaw, 93 Zwirki Wigury St, 02-089, Warsaw, Poland

    Anna Modrak-Wójcik, Aneta Kirilenko, David Shugar & Borys Kierdaszuk

  2. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5a Pawinskiego St, 02-106, Warsaw, Poland

    David Shugar

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  1. Anna Modrak-Wójcik
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  2. Aneta Kirilenko
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Correspondence to Borys Kierdaszuk.

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Modrak-Wójcik, A., Kirilenko, A., Shugar, D. et al. Role of ionization of the phosphate cosubstrate on phosphorolysis by purine nucleoside phosphorylase (PNP) of bacterial (E. coli) and mammalian (human) origin. Eur Biophys J 37, 153–164 (2008). https://doi.org/10.1007/s00249-007-0205-8

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