Thermal stability of Escherichia coli ribonuclease HI and its active site mutants in the presence and absence of the Mg2+ ion. Proposal of a novel catalytic role for Glu48.
Escherichia coli ribonuclease HI, which requires divalent cations (Mg2+ or Mn2+) for activity, was thermostabilized by 2.6-3.0 kcal/mol in the presence of the Mg2+, Mn2+, or Ca2+ ion, probably because the negative charge repulsion around the active site was canceled upon the binding of these metal ions. The dissociation constants were determined to be 0.71 mM for Mg2+, 0.035 mM for Mn2+, and 0.16 mM for Ca2+. Likewise, various active site mutants at Asp10, Glu48, Asp70, or Asp134 were thermostabilized by 0.4-3.0 kcal/mol in the presence of the Mg2+ ion, suggesting that this ion binds to these mutant proteins as well. The dissociation constants of Mg2+ were determined to be 9.8 mM for D10N, 1.1 mM for E48Q, 18.8 mM for D70N, and 1.8 mM for D134N. Thus, the mutation of Asp10 or Asp70 to Asn considerably impairs the Mg2+ binding, whereas the mutation of Glu48 to Gln or Asp134 to Asn does not. Comparison of the thermal stability of the mutant proteins with that of the wild-type protein in the absence of the Mg2+ ion suggests that the negative charge repulsion between Asp10 and Asp70 is responsible for the binding of the metal cofactor. Glu48 may be required to anchor a water molecule, which functions as a general acid.
Kanaya
S
S
Protein Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565, Japan. kanaya@chem.eng.osaka-u.ac.jp
Oobatake
M
M
Liu
Y
Y
D000838:Anions; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002412:Cations; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates / Q000737:chemistry; D006358:Hot Temperature; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D016914:Ribonuclease H / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D000838:Anions; D001426:Bacterial Proteins; D002412:Cations; D005971:Glutamates; D016914:Ribonuclease H; C078856:ribonuclease HI; D008274:Magnesium