Understanding Ion‐specific “Hofmeister” Effects in Enzyme Catalysis through using RNase A as a Paradigm Model

Biophysical studies in the last two decades have clearly demonstrated that salts affect biomolecules in an ion-specific manner (i. e., Hofmeister Effects). Studies performed upon such diverse biological processes such as protein folding, protein precipitation, protein coacervation and phase separation, and protein oligomerization, have all shown that this ion specificity is directly related to how individual ions interact with biomolecular surfaces. Interestingly, although ion-specific effects upon enzyme catalytic processes are well-known in the literature, a molecular level description of these effects has not yet been made available. For example, it is not clear whether ion-specific effects observed in enzyme catalysis are directly related to how ions modulate the enzyme's folding free energy, or not. This work attempts to address this need by investigating ion-specific effects upon the enzymatic activity and folding free energy of a well-characterized enzyme system, Ribonuclease A (RNase A). To this end we have developed a robust framework to analyze and quantify ion-specific effects upon the RNase A catalyzed phosphate ring opening reaction of cCMP (Cytidine 2':3'-cyclic monophosphate monosodium salt). Our studies show that both the folding thermodynamics and the Michaelis-Menten kinetic parameters of this enzyme show ion-specific salt dependence. However, even through salt addition affects the folding free energy and enzyme catalysis of RNase A in an ion-specific manner, these effects are not necessarily directly related to each other. Ion-specific effects observed in protein folding reflects mostly how an individual ion interacts with the overall protein surface; while alternatively, ion-specific effects on enzyme activity indicate how a given ion interacts with the enzyme active site surface or alternatively, how ions interact with the substrate molecule as represented by changes in the substrate thermodynamic activity coefficient.