A catalytically deficient active site variant of PvuII endonuclease binds Mg(II) ions.
In efforts to understand the mechanisms of many nucleic acid enzymes, the first site-directed mutations are made at conserved acidic active residues. Almost without exception, the low or null activities of the resulting variants are attributed to the importance of the acidic residue(s) to the ligation of required metal ions. Using (25)Mg NMR spectroscopy as a direct probe of metal ion binding and the homodimeric PvuII restriction endonuclease as a model system, this interpretation is examined and clarified. Our results indicate that Mg(II) binds wild-type PvuII endonuclease in the absence of DNA with a K(d,app) of 1.9 mM. Hill analysis yields an n(H) coefficient of 1.4, a value consistent with the binding of more than one Mg(II) ion per monomer active site. Variable pH studies indicate that two ionizable groups are responsible for Mg(II) binding by wild-type PvuII endonuclease near physiological pH. The pK(a,app) for these ionizations is 6.7, a value which is unusual for acidic residues but consistent with data obtained for critical groups in MunI endonuclease and a number of other hydrolases. To assign residues critical to ligating Mg(II), binding measurements were performed on the low activity catalytic site mutants E68A and D58A. As expected, E68A binds Mg(II) ions very weakly (K(d,app) approximately 40 mM), implicating Glu68 as critical to Mg(II) binding. Interestingly, while D58A has only residual specific activity, it retains an affinity for Mg(II) with a K(d,app) of 3.6 mM and exhibits a Hill coefficient of 0.7. Moreover, in this variant, multiple ionizable groups with pK(a,app) of 7.2 are involved in Mg(II) binding, suggesting a shuffling of Mg(II) ligands in the active site. These data indicate that Asp58 is important for the critical positioning of metal ion(s) required for catalysis.
Dupureur
C M
CM
Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA. cdub@tamu.edu
Conlan
L H
LH
D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014644:Genetic Variation; D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D008024:Ligands; D008274:Magnesium / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017186:Titrimetry
D002413:Cations, Divalent; D007554:Isotopes; D008024:Ligands; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium; D002118:Calcium