Abstract
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.
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Abbreviations
- cCMB:
-
Cytidine-2′,3′-cyclic borate
- cCMP:
-
Cytidine-2′,3′-cyclic phosphate
- CMB:
-
3′-Deoxycytidine-2′-monoborate ester
- 2′-CMP:
-
Cytidine-2′-monophosphate
- 3′-CMP:
-
Cytidine-3′-monophosphate
- γGT:
-
γ-Glutamyl transpeptidase
- HEPES:
-
N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid
- PDB:
-
Protein Data Bank
- RNase A:
-
Ribonuclease A
- Tris:
-
Tris(hydroxymethyl)aminomethane
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Acknowledgements
The authors are grateful to Yi-chien Lee for providing a preprint of his study of the interaction of 3′-CMP with RNase A. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.
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Gabel, S.A., London, R.E. Ternary borate–nucleoside complex stabilization by ribonuclease A demonstrates phosphate mimicry. J Biol Inorg Chem 13, 207–217 (2008). https://doi.org/10.1007/s00775-007-0311-1
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DOI: https://doi.org/10.1007/s00775-007-0311-1