The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI ‫ס‬ 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI ‫ס‬ 6.4 and a 5K variant (3K + D25K, E74K) with a pI ‫ס‬ 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.

A ribonuclease from a callus cell culture of Panax ginseng C.A. Mey strain R1 was isolated. A pure protein with an apparent molecular mass of 18 kDa was obtained. The N-terminal sequences of the protein and of the C-terminal CNBr peptide were determined. No homology with other ribonucleases was found, but there was 60-70% sequence identity with two intracellular pathogenesis-related (IPR) proteins from parsley, indicating that not only these two proteins, but also homologous IPR proteins identified in other plant species are ribonucleases.

Parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase in the pH region 2-6 have been determined. The barnase heat denaturation (pH 2.8-5.5) proceeds according to the "all-or-none" principle. Barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 degrees C at pH 5 to 7 degrees C at pH 3. Enthalpy values of barnase and binase denaturation coincide only at pH 4.5-5.5, but as far as pH decreases the barnase denaturation enthalpy decreases significantly and in this respect it differs from binase. The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the barnase Trp94 by the His 18 residue, absent in binase. Secondary structures of both native and denaturated proteins also do not differ. Some differences in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moments distribution, have been found.

No abstract

To elucidate the functional role of some residues in the active site of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant His""Glu is 2.0-2.7% of that for native enzyme. The decrease in activity is determined mainly by the decrease in molecular rate constant k,,, with almost unchanged affinity of the enzyme for the substrate, characterized by KM. This is the expected result if His"' acts as an general acid, donating a proton to the leaving group on cleavage of a phosphodiester bond. The replacement of Lysz6 by Ala causes a reduction in the enzyme activity to 13-33%, depending on the substrate. The activity decreases are due to changes in both k,, and KM for poly(1) and poly(A) but in t,, alone for GpA. In the latter case the effect is far less than that seen in the homologous mutation in the closely related enzyme, bamase.