Effects of Salt and pH on Binding and Catalysis by Ribonuclease A

Salt concentration and pH have dramatic effects on enzymatic catalysis. A quantitative description on these effects is important to elucidate the energetics and mechanisms of catalysis. Here, the effects of salt concentration and pH on binding and catalysis are analyzed with Ribonuclease A (RNase A) as a model system. 11 The effects of pH and mutagenesis on the stability of RNase A-nucleic acids complexes were studied by biophysical methods (Chapter 2). The thermodynamic information indicates that the active-site histidine residues make a significant contribution to nucleic acid binding as well as to catalytic turnover. The origin of RNase A "inactivation" at low salt concentration is scrutinized by kinetic studies (Chapter 3). The results show that inactivation results from the inhibition of RNase A activity by low-level contaminants from common buffer solutions. The salt effect on RNase A catalysis is analyzed with a novel quantitative model (Chapter 4). This model is applied successfully to explain salt-rate profiles of RNase A. The energetics of RNase A catalysis is re-examined by the effects of pH, salt concentration, and solvent isotope, which show that substrate association limits catalysis by RNase A (Chapter 5). By protein complementation with H12A RNase A and Hl19A RNase A, dimerization by RNase A is detected and analyzed, providing clues for the mechanism of dimer evolution (Chapter 6). Finally, adjacent cysteine residues were introduced into RNase A as a redox switch (Chapter 7). The catalytic activity of the RNase A variant is regulated by the redox potential of the medium.