Contribution of the active site histidine residues of ribonuclease A to nucleic acid binding

His12 and His119 are critical for catalysis of RNA cleavage by ribonuclease A (RNase A). Substitution of either residue with an alanine decreases the value of k(cat)/K-M by more than 10(4)-fold. His12 and His119 are proximal to the scissile phosphoryl group of an RNA substrate in enzyme-substrate co mplexes. Here, the role of these active site histidines in RNA binding was investigated by monitoring the effect of mutagenesis and pH on the stabilit y of enzyme-nucleic acid complexes. X-ray diffraction analysis of the H12A and H119A variants at a resolution of 1.7 and 1.8 Angstrom, respectively, s hows that the amino acid substitutions do not perturb the overall structure of the variants. Isothermal titration calorimetric studies on the complexa tion of wild-type RNase A and the variants with 3 ' -UMP at pH 6.0 show tha t His12 and His119 contribute 1.4 and 1.1 kcal/mol to complex stability, re spectively. Determination of the stability of the complex of wild-type RNas e A and 6-carboxyfluorescein similar tod(AUAA) at varying pHs by fluorescen ce anisotropy shows that the stability increases by 2.4 kcal/mol as the pH decreases from 8.0 to 4,0, At pH 4,0, replacing His12 with an alanine resid ue decreases the stability of the complex with 6-carboxyfluorescein similar tod(AUAA) by 2.3 kcal/mol. Together, these structural and thermodynamic da ta provide the first thorough analysis of the contribution of histidine res idues to nucleic acid binding.