JBIC Journal of Biological Inorganic Chemistry

, Volume 13, Issue 2, pp 207–217

Ternary borate–nucleoside complex stabilization by ribonuclease A demonstrates phosphate mimicry

Original Paper


Phosphate esters play a central role in cellular energetics, biochemical activation, signal transduction and conformational switching. The structural homology of the borate anion with phosphate, combined with its ability to spontaneously esterify hydroxyl groups, suggested that phosphate ester recognition sites on proteins might exhibit significant affinity for nonenzymatically formed borate esters. 11B NMR studies and activity measurements on ribonuclease A (RNase A) in the presence of borate and several cytidine analogs demonstrate the formation of a stable ternary RNase A·3′-deoxycytidine–2′-borate ternary complex that mimics the complex formed between RNase A and a 2′-cytidine monophosphate (2′-CMP) inhibitor. Alternatively, no slowly exchanging borate resonance is observed for a ternary RNase A, borate, 2′-deoxycytidine mixture, demonstrating the critical importance of the 2′-hydroxyl group for complex formation. Titration of the ternary complex with 2′-CMP shows that it can displace the bound borate ester with a binding constant that is close to the reported inhibition constant of RNase A by 2′-CMP. RNase A binding of a cyclic cytidine-2′,3′-borate ester, which is a structural homolog of the cytidine-2′,3′-cyclic phosphate substrate, could also be demonstrated. The apparent dissociation constant for the cytidine-2′,3′-borate·RNase A complex is 0.8 mM, which compares with a Michaelis constant of 11 mM for cytidine-2′,3′-cyclic phosphate at pH 7, indicating considerably stronger binding. However, the value is 1,000-fold larger than the reported dissociation constant of the RNase A complex with uridine–vanadate. These results are consistent with recent reports suggesting that in situ formation of borate esters that mimic the corresponding phosphate esters supports enzyme catalysis.


Nuclear magnetic resonance Nucleic acid Ribonuclease A Borate 11B NMR 



Cytidine-2′,3′-cyclic borate


Cytidine-2′,3′-cyclic phosphate


3′-Deoxycytidine-2′-monoborate ester






γ-Glutamyl transpeptidase


N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid


Protein Data Bank

RNase A

Ribonuclease A




  1. 1.
    Bertagnolli BL, Hanson JB (1973) Plant Physiol 52:431–435PubMedGoogle Scholar
  2. 2.
    Gresser MJ (1981) J Biol Chem 256:5981–5983PubMedGoogle Scholar
  3. 3.
    Moore SA, Moennich DMC, Gresser MJ (1983) J Biol Chem 258:6266–6271PubMedGoogle Scholar
  4. 4.
    Borah B, Chen CW, Egan W, Miller M, Wlodawer A, Cohen JS (1985) Biochemistry 24:2058–2067PubMedCrossRefGoogle Scholar
  5. 5.
    Rehder D, Holst H, Quaas R, Hinrichs W, Hahn U, Saenger W (1989) J Inorg Biochem 37:141–150PubMedCrossRefGoogle Scholar
  6. 6.
    Georgalis Y, Zouni A, Hahn U, Saenger W (1991) Biochim Biophys Acta 1118:1–5PubMedGoogle Scholar
  7. 7.
    Krauss M, Basch H (1992) J Am Chem Soc 114:3630–3634CrossRefGoogle Scholar
  8. 8.
    Wladkowski BD, Svensson LA, Sjolin L, Ladner JE, Gilliland GL (1998) J Am Chem Soc 120:5488–5498CrossRefGoogle Scholar
  9. 9.
    Leon-Lai CH, Gresser MJ, Tracey AS (1996) Can J Chem 74:38–48CrossRefGoogle Scholar
  10. 10.
    Zhang M, Zhou M, VanEtten RL, Stauffacher CV (1997) Biochemistry 36:15–23PubMedCrossRefGoogle Scholar
  11. 11.
    Lima CD, Klein MG, Hendrickson WA (1997) Science 278:286–290PubMedCrossRefGoogle Scholar
  12. 12.
    Rupert PB, Massey AP, Sigurdsson ST, Ferre-D’Amare AR (2002) Science 298:1421–1424PubMedCrossRefGoogle Scholar
  13. 13.
    Huyer G, Liu S, Kelly J, Moffat J, Payette P, Kennedy B, Tsaprailis G, Gresser MJ, Ramachandran C (1997) J Biol Chem 272:843–851PubMedCrossRefGoogle Scholar
  14. 14.
    Sternweis PC, Gilman AG (1982) Proc Natl Acad Sci USA 79:4888–4891PubMedCrossRefGoogle Scholar
  15. 15.
    Maruta S., Henry GD, Sykes BD, Ikebe M (1993) J Biol Chem 268:7093–7100PubMedGoogle Scholar
  16. 16.
    Sondek J, Lambright DG, Noel JP, Hamm HE, Sigler PB (1994) Nature 372:276–279PubMedCrossRefGoogle Scholar
  17. 17.
    Fisher AJ, Smith CA, Thoden JB, Smith R, Sutoh K, Holden HM, Rayment I (1995) Biochemistry 34:8960–8972PubMedCrossRefGoogle Scholar
  18. 18.
    Dominguez R, Freyzon Y, Trybus KM, Cohen C (1998) Cell 94:559–571PubMedCrossRefGoogle Scholar
  19. 19.
    Coureux P-D, Sweeney HL, Houdusse A (2004) EMBO J 23:4527–4537PubMedCrossRefGoogle Scholar
  20. 20.
    Smith KW, Johnson SL (1976) Biochemistry 15:560–565PubMedCrossRefGoogle Scholar
  21. 21.
    Kim DH, Marbois BN, Faull KF, Eckhert CD (2003) J Mass Spectrom 38:632–640PubMedCrossRefGoogle Scholar
  22. 22.
    Kim DH, Faull KF, Norris AJ, Eckhert CD (2004) J Mass Spectrom 39:743–751PubMedCrossRefGoogle Scholar
  23. 23.
    Sugiyama M, Hong Z, Whalen LJ, Greenberg WA, Wong C-H (2006) Adv Synth Catal 348:2555–2559CrossRefGoogle Scholar
  24. 24.
    Tate SS, Meister A (1978) Proc Natl Acad Sci USA 75:4806–4809PubMedCrossRefGoogle Scholar
  25. 25.
    London RE, Gabel SA (2001) Arch Biochem Biophys 385:250–258PubMedCrossRefGoogle Scholar
  26. 26.
    London RE, Gabel SA (2002) Biochemistry 41:5963–5967PubMedCrossRefGoogle Scholar
  27. 27.
    Transue TR, Krahn JM, Gabel SA, DeRose F, London RE (2004) Biochemistry 43:2829–2839PubMedCrossRefGoogle Scholar
  28. 28.
    Babine RE, Rynkiewicz MJ, Jin L, Abdel-Meguid SS (2004) Lett Drug Des Discov 1:35–44CrossRefGoogle Scholar
  29. 29.
    Raines RT (1998) Chem Rev 98:1045–1065PubMedCrossRefGoogle Scholar
  30. 30.
    Herries DG, Mathias AP, Rabin BR (1962) Biochem J 85:127–134PubMedGoogle Scholar
  31. 31.
    Crook EM, Mathias AP, Rabin BR (1960) Biochem J 74:234–238PubMedGoogle Scholar
  32. 32.
    Coddington JM, Taylor MJ (1989) J Coord Chem 20:27–38Google Scholar
  33. 33.
    Baker WR, Kintanar A (1996) Arch Biochem Biophys 327:189–199PubMedCrossRefGoogle Scholar
  34. 34.
    Babcock L, Pizer R (1980) Inorg Chem 19:56–61CrossRefGoogle Scholar
  35. 35.
    Hahn U, Desai-Hahn R, Ruterjans H (1985) Eur J Biochem 146:705–712PubMedCrossRefGoogle Scholar
  36. 36.
    El Harrous M, Parody-Morreale A (1997) Anal Biochem 254:96–108CrossRefGoogle Scholar
  37. 37.
    Perlman ME, Davis DG, Koszalka GW, Tuttle JV, London RE (1994) Biochemistry 33:7547–7559PubMedCrossRefGoogle Scholar
  38. 38.
    Lisgarten JN, Gupta V, Maes D, Wyns L, Zegers I, Palmer RA, Dealwis CG, Aguilar CF, Hemmings AM (1993) Acta Crystallogr Sect D 49:541–547CrossRefGoogle Scholar
  39. 39.
    Zegers I, Maes D, Dao-Thi M-H, Poortmans F, Palmer R, Wyns L (1994) Protein Sci 3:2322–2339PubMedCrossRefGoogle Scholar
  40. 40.
    Pizer R, Selzer R (1984) Inorg Chem 23:3023–3026CrossRefGoogle Scholar
  41. 41.
    Weser U (1967) Z Naturforsch B 22:457–458PubMedGoogle Scholar
  42. 42.
    Chapelle S, Verchere J-F (1988) Tetrahedron 44:4469–4482CrossRefGoogle Scholar
  43. 43.
    Antonov AK, Ivanina TV, Berezin IV, Martinek K (1968) Dokl Acad Nauk SSSR 183:1435–1438Google Scholar
  44. 44.
    Yonetani T, Theorell H (1964) Arch Biochem Biophys 106:243–251PubMedCrossRefGoogle Scholar
  45. 45.
    Transue TR, Gabel SA, London RE (2006) Bioconj Chem 17:300–308CrossRefGoogle Scholar
  46. 46.
    Oki T, Yoshimoto A, Sato S, Takamatsu A (1975) Biochim Biophys Acta 410:262–272PubMedGoogle Scholar
  47. 47.
    Reddi KK, Dreiling DA (1982) Clin Biochem 15:109–112PubMedCrossRefGoogle Scholar
  48. 48.
    Sharp KA, Friedman RA, Misra V, Hecht J, Honig B (1995) Biopolymers 36:245–262PubMedCrossRefGoogle Scholar
  49. 49.
    Bauer C-A, Pettersson G (1974) Eur J Biochem 45:473–477PubMedCrossRefGoogle Scholar
  50. 50.
    Dreyer MK, Schulz GE (1996) J Mol Biol 259:458–466PubMedCrossRefGoogle Scholar
  51. 51.
    Reuter W, Wiegand G, Huber R, Than ME (1999) Proc Natl Acad Sci USA 96:1363–1368PubMedCrossRefGoogle Scholar

Copyright information

© SBIC 2007

Authors and Affiliations

  1. 1.MR-01, Laboratory of Structural BiologyNational Institute of Environmental Health Sciences, NIHResearch Triangle ParkUSA

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