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Site-Specific Protonation Kinetics of Acidic Side Chains in Proteins Determined by pH-Dependent Carboxyl 13C NMR Relaxation

Proton-transfer dynamics plays a critical role in many biochemical processes, such as proton pumping across membranes and enzyme catalysis. The large majority of enzymes utilize acid–base catalysis and proton-transfer mechanisms, where the rates of proton transfer can be rate limiting for the overal...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2015-03, Vol.137 (8), p.3093-3101
Main Authors: Wallerstein, Johan, Weininger, Ulrich, Khan, M. Ashhar I, Linse, Sara, Akke, Mikael
Format: Article
Language:English
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Summary:Proton-transfer dynamics plays a critical role in many biochemical processes, such as proton pumping across membranes and enzyme catalysis. The large majority of enzymes utilize acid–base catalysis and proton-transfer mechanisms, where the rates of proton transfer can be rate limiting for the overall reaction. However, measurement of proton-exchange kinetics for individual side-chain carboxyl groups in proteins has been achieved in only a handful of cases, which typically have involved comparative analysis of mutant proteins in the context of reaction network modeling. Here we describe an approach to determine site-specific protonation and deprotonation rate constants (k on and k off, respectively) of carboxyl side chains, based on 13C NMR relaxation measurements as a function of pH. We validated the method using an extensively studied model system, the B1 domain of protein G, for which we measured rate constants k off in the range (0.1–3) × 106 s–1 and k on in the range (0.6–300) × 109 M–1 s–1, which correspond to acid–base equilibrium dissociation constants (K a) in excellent agreement with previous results determined by chemical shift titrations. Our results further reveal a linear free-energy relationship between log k on and pK a, which provides information on the free-energy landscape of the protonation reaction, showing that the variability among residues in these parameters arises primarily from the extent of charge stabilization of the deprotonated state by the protein environment. We find that side-chain carboxyls with extreme values of k off or k on are involved in hydrogen bonding, thus providing a mechanistic explanation for the observed stabilization of the protonated or deprotonated state.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja513205s