THERMAL-STABILITY OF ESCHERICHIA-COLI RIBONUCLEASE HI AND ITS ACTIVE-SITE MUTANTS IN THE PRESENCE AND ABSENCE OF THE MG2- PROPOSAL OF A NOVEL CATALYTIC ROLE FOR GLU(48)( ION )

Escherichia coli ribonuclease HI, which requires divalent cations (Mg2 + or Mn2+) for activity, was thermostabilized by 2.6-3.0 kcal/mol in t he presence of the Mg2+, Mn2+, or Ca2+ ion, probably because the negat ive charge repulsion around the active site was canceled upon the bind ing 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+. Likewis e, various active site mutants at Asp(10), Glu(48), Asp(70), or Asp(13 4) were thermostabilized by 0.4-3.0 kcal/mol in the presence of the Mg 2+ ion, suggesting that this ion binds to these mutant proteins as wel l. 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 Asp(10) or Asp(70) to Asn considerably impairs the Mg2 + binding, whereas the mutation of Glu(48) to Gln or Asp(134) to Asn d oes not. Comparison of the thermal stability of the mutant proteins wi th that of the wild-type protein in the absence of the Mg2+ ion sugges ts that the negative charge repulsion between Asp10 and Asp(70) is res ponsible for the binding of the metal cofactor. Glu(48) may be require d to anchor a water molecule, which functions as a general acid.