JBIC Journal of Biological Inorganic Chemistry

, Volume 12, Issue 4, pp 557–569

Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis

Authors

  • Grigorios A. Papadakos
    • Department of Chemistry and BiochemistryUniversity of Missouri-St Louis
  • Horacio Nastri
    • New England Biolabs
    • EMD Lexigen Research Center
  • Paul Riggs
    • New England Biolabs
    • Department of Chemistry and BiochemistryUniversity of Missouri-St Louis
Original Paper

DOI: 10.1007/s00775-007-0209-y

Cite this article as:
Papadakos, G.A., Nastri, H., Riggs, P. et al. J Biol Inorg Chem (2007) 12: 557. doi:10.1007/s00775-007-0209-y

Abstract

The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 Å from both metal ions in the enzyme–substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H–15N heteronuclear single quantum coherence spectra of enzyme–Ca(II) and enzyme–Ca(II)–DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.

Keywords

NucleaseEnzyme mechanismMetal ionsDNA

Abbrevations

HEPES

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

HSQC

Heteronuclear single quantum coherence

Tris

Tris(hydroxymethyl)aminomethane

TROSY

Transverse relaxation optimized spectroscopy

WT

Wild type

Copyright information

© SBIC 2007