Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis
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 A 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.
Available from: Sasa Bjelic
[Show abstract] [Hide abstract]
ABSTRACT: Elucidation of the energetic principles of binding affinity and specificity is a central task in many branches of current sciences: biology, medicine, pharmacology, chemistry, material sciences, etc. In biomedical research, integral approaches combining structural information with in-solution biophysical data have proved to be a powerful way toward understanding the physical basis of vital cellular phenomena. Isothermal titration calorimetry (ITC) is a valuable experimental tool facilitating quantification of the thermodynamic parameters that characterize recognition processes involving biomacromolecules. The method provides access to all relevant thermodynamic information by performing a few experiments. In particular, ITC experiments allow to by-pass tedious and (rarely precise) procedures aimed at determining the changes in enthalpy and entropy upon binding by van't Hoff analysis. Notwithstanding limitations, ITC has now the reputation of being the ''gold standard'' and ITC data are widely used to validate theoretical predictions of thermodynamic parameters, as well as to benchmark the results of novel binding assays. In this paper, we discuss several publications from 2007 reporting ITC results. The focus is on applications in biologically oriented fields. We do not intend a comprehensive coverage of all newly accumulated information. Rather, we emphasize work which has captured our attention with originality and far-reaching analysis, or else has provided ideas for expanding the potential of the method.
Journal of Molecular Recognition 09/2008; 21(5):289-312. DOI:10.1002/jmr.909 · 2.15 Impact Factor
Available from: Cynthia Dupureur
[Show abstract] [Hide abstract]
ABSTRACT: Most nucleases rely on divalent cations as cofactors to catalyze the hydrolysis of nucleic acid phosphodiester bonds. Here both equilibrium and kinetic experiments are used to test recently proposed models regarding the metal ion dependence of product release and the degree of cooperativity between metal ions bound in the active sites of the homodimeric PvuII endonuclease. Equilibrium fluorescence anisotropy studies indicate that product binding is dramatically weakened in the presence of metal ions. Pre-steady state kinetics indicate that product release is at least partially rate limiting. Steady state and pre-steady state data fit best to models in which metals remain bound to the enzyme after the release of product. Finally, analysis of cooperative and independent binding models for metal ions indicates that single turnover kinetic data are consistent with little to no positive cooperativity between the two metal ions binding each active site.
Archives of Biochemistry and Biophysics 02/2009; 483(1):1-9. DOI:10.1016/j.abb.2009.01.001 · 3.02 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Much effort has been directed at understanding the roles of metal ions in catalyzing the hydrolysis of phosphodiester bonds of nucleic acids. Nucleases are metalloenzymes that have a wide variety of active site motifs and that contain a variety of different metal ions. This property has made it difficult to propose a simple mechanism for these enzymes. Therefore, design and synthesis of metal complexes, which can mediate phosphodiester bond cleavage via hydrolytic pathways, are of important significance in elucidation of the catalytic mechanisms for the natural nucleases and in development of the biomacromolecule-targeted drugs. Recent progress has extended to the design of synthetic multinuclear metallonucleases containing two or more Fe(III), Zn(II), Cu(II), Co(II/III), or Ln(III/IV) ions. The ligands in these complexes include natural and nonnatural organic molecules, i.e., mainly benzimidazolyl- and pyridyl-based organic molecules, azamacrocyclic and aminocarboxylic derivatives, and their conjugates to polypeptides or oligonucleotides. The purpose of this perspective is to highlight: (1) the differences in structure and composition between natural and synthetic multinuclear metallonucleases; (2) the design strategies of synthetic multinuclear metallonucleases; (3) the relationship between the structures and nucleolytic activities of synthetic multinuclear metallonucleases; and (4) the cooperativities between metal sites, and between metal sites and ligands in the courses of phospodiester linkage hydrolysis. A comparison illustrates unifying themes in the catalysis of phosphodiester linkage hydrolysis by natural and synthetic multinuclear metallonucleases. Indeed, there are features that converge about the chemistry that provides insight into how changes in metal ions and ligands of both natural and synthetic metallonucleases may lead to the same overall outcome of phosphodiester backbone cleavage. In addition, we will also discuss the solvation effect of synthetic multinuclear metallonucleases and the challenges that should be faced toward the development of synthetic multinuclear metallonucleases with DNA sequence or structure selectivity by applying the principles of coordination and enzymatic chemistry.
Dalton Transactions 02/2009; 2(2):227-39. DOI:10.1039/b811616d · 4.20 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.