Protonation kinetics in proteins at basic pH determined by pH-dependent NMR relaxation reveal the entire relationship between kinetics and pKa values

Abstract

Ionizable amino acid side chains in proteins undergo constant protonation and deprotonation reactions. These proton exchange dynamics are a fundamental feature of proteins and their electrostatic character, as well as the basis for many biological processes, such as general acid–base enzyme catalysis. Such dynamics have been measured in a site-specific way for aspartates, glutamates, and histidines by pH-dependent NMR relaxation experiments. Linear free-energy relationships between kinetic and thermodynamic parameters have been established that allow the description of proton-mediated proton exchange at low to neutral pH. Here, we complement the picture by determining the proton exchange kinetics of lysine and tyrosine side chains at basic pH. They display matching linear free-energy relationships that enable the description of hydroxide-mediated proton exchange at high pH. The underlying maximal second-order rate constants are approximately a factor of 40 higher for hydronium association compared to hydroxide dissociation. These combined findings provide a general framework for describing protonation kinetics, allowing for the prediction of protonation and deprotonation rate constants for ionizable groups with all possible pKa values across the entire pH range.