Protonation linked equilibria and apparent affinity constants: the thermodynamic profile of the α-chymotrypsin–proflavin interaction
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Protonation/deprotonation equilibria are frequently linked to binding processes involving proteins. The presence of these thermodynamically linked equilibria affects the observable thermodynamic parameters of the interaction (K obs, ΔH obs 0 ). In order to try and elucidate the energetic factors that govern these binding processes, a complete thermodynamic characterisation of each intrinsic equilibrium linked to the complexation event is needed and should furthermore be correlated to structural information. We present here a detailed study, using NMR and ITC, of the interaction between α-chymotrypsin and one of its competitive inhibitors, proflavin. By performing proflavin titrations of the enzyme, at different pH values, we were able to highlight by NMR the effect of the complexation of the inhibitor on the ionisable residues of the catalytic triad of the enzyme. Using ITC we determined the intrinsic thermodynamic parameters of the different equilibria linked to the binding process. The possible driving forces of the interaction between α-chymotrypsin and proflavin are discussed in the light of the experimental data and on the basis of a model of the complex. This study emphasises the complementarities between ITC and NMR for the study of binding processes involving protonation/deprotonation equilibria.
KeywordsNuclear Magnetic Resonance Isothermal Titration Calorimetry Protonation State Affinity Constant Titratable Group
G.B. thanks the Belgian FRIA for a PhD fellowship and the Foundation Wiener-Anspach for a post-doctoral fellowship. C.R. thanks the Wellcome Trust for a “Value in People” award. The authors acknowledge Professor Jacques Reisse for very helpful discussions. This work was supported by the Belgian FNRS (LEA CNRS-FNRS), the “Communauté Française de Belgique” (ARC 2002–2007).
- Baker BM, Murphy KP (1996) Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry. Biophys J 71:2049–2055Google Scholar
- Bernhard SA, Lee BF, Tashjian ZH (1966) On the interaction of the active side of alpha-chymotrypsin with chromophores: proflavin binding and enzyme conformation during catalysis. J Mol Biol 18:405–420Google Scholar
- Boutonnet NS, Rooman MJ, Ochagavia M-E, Richelle J, Wodak SJ (1995) Optimal protein structure alignments by multiple linkage clustering: application to distantly related proteins. Protein Eng 8:647–662Google Scholar
- Havsteen BH (1967) Kinetics of the two-step interaction of chymotrypsin with proflavine. J Biol Chem 242:769–771Google Scholar
- Shiao DDF, Sturtevant JM (1969) Calorimetric investigations of the binding of inhibitors to α-chymotrypsin. I. Enthalpy of dilution of α-chymotrypsin and of proflavine, and the enthalpy of binding of indole, N-acetyl-D-tryptophan, and proflavine to α-chymotrypsin. Biochemistry 8:4910–4917CrossRefGoogle Scholar
- Tulinsky A, Blevins RA (1987) Structure of a tetrahedral transition state complex of alpha-chymotrypsin dimer at 1.8-Å resolution. J Biol Chem 262:7737–7743Google Scholar
- Waelbroeck M (1982) The pH dependence of insulin binding. A quantitative study. J Biol Chem 257:8284–8291Google Scholar
- Zhong S, Haghjoo K, Kettner C, Jordan F (1995) Proton magnetic resonance studies of the active center histidine of chymotrypsin complexed to peptideboronic acids: solvent accessibility to the Nδ and Nε sites can differentiate slow-binding and rapidly reversible inhibitors. J Am Chem Soc 117:7048–7055CrossRefGoogle Scholar