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

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.

Keywords

Nuclear magnetic resonance Nucleic acid Ribonuclease A Borate 11B NMR 

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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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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