Characterization of pKa values and titration shifts in the cytotoxic ribonuclease α-sarcin by NMR.: Relationship between electrostatic interactions, structure, and catalytic function

The electrostatic behavior of titrating groups in alpha-sarcin was investigated using H-1 NMR spectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in the molecule were determined over the pH range of 3.0-8.5. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the unambiguous determination of pK(a) values for all glutamic acid and histidine residues, as well as for the C-terminal carboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalytically relevant histidines, His50 and His137, and glutamic acid, Glu96, in the alpha-sarcin-2'-GMP complex were also determined. The pK(a) values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamic acids, and four histidines) were found to be close to their normal values. On the other hand, a number of side chain groups, including those in the active center, showed pK(a) values far from their intrinsic values. Thus, the pK(a) values for active site residues His50, Glu96, and His137 were 7.7, 5.2, and 5.8 in the free enzyme and 7.6, similar to 4.8, and 6.8 in the alpha-sarcin-2'-GMP complex, respectively. The pK(a) values and the activity profile against ApA, as a function of pH, are in agreement with the proposed enzymatic mechanism (in common with RNase T1 and the family of the microbial ribonucleases), in which Glu96 and His137 act as a general base and general acid, respectively. In almost all microbial ribonucleases, a Phe-His interaction is present, which affects the pK(a) of one of the His residues at the active site (His137). The absence of this interaction in alpha-sarcin would explain the lower pK(a) value of this His residue, and provides an explanation for the decreased RNase activity of this protein as compared to those of other microbial ribonucleases.