Quantitative Analysis of the Effect of Salt Concentration on Enzymatic Catalysis

Publisher Website
Google Scholar
Abstract
Like pH, salt concentration can have a dramatic effect on enzymatic catalysis. Here, a general equation is derived for the quantitative analysis of salt−rate profiles: kcat/KM = (kcat/KM)MAX/[1 + ([Na+]/KNa+)n‘], where (kcat/KM)MAX is the physical limit of kcat/KM, KNa+ is the salt concentration at which kcat/KM = (kcat/KM)MAX/2, and −n‘ is the slope of the linear region in a plot of log(kcat/KM) versus log [Na+]. The value of n‘ is of special utility, as it reflects the contribution of Coulombic interactions to the uniform binding of the bound states. This equation was used to analyze salt effects on catalysis by ribonuclease A (RNase A), which is a cationic enzyme that catalyzes the cleavage of an anionic substrate, RNA, with kcat/KM values that can exceed 109 M-1 s-1. Lys7, Arg10, and Lys66 comprise enzymic subsites that are remote from the active site. Replacing Lys7, Arg10, and Lys66 with alanine decreases the charge on the enzyme as well as the value of n‘. Likewise, decreasing the number of phosphoryl groups in the substrate decreases the value of n‘. Replacing Lys41, a key active-site residue, with arginine creates a catalyst that is limited by the chemical conversion of substrate to product. This change increases the value of n‘, as expected for a catalyst that is more sensitive to changes in the binding of the chemical transition state. Hence, the quantitative analysis of salt−rate profiles can provide valuable insight into the role of Coulombic interactions in enzymatic catalysis.