Analysis of dynamics and mechanism of ligand binding toartocarpus integrifolia agglutinin. A ¹³C and ¹⁹F NMR study

Abstract

Binding of ¹³C-labeled N-acetylgalactosamine (¹³C-GalNAc) and N-trifluoroacetylgalactosamine (¹⁹F-GalNAc) to Artocarpus integrifolia agglutinin has been studied using ¹³C and ¹⁹F nuclear magnetic resonance spectroscopy, respectively. Binding of these saccharides resulted in broadening of the resonances, and no change in chemical shift was observed, suggesting that the α- and β-anomers of ¹³C-GalNAc and ¹⁹F-GalNAc experience a magnetically equivalent environment in the lectin combining site. The α- and β-anomers of ¹³C-GalNAc and ¹⁹F-GalNAc were found to be in slow exchange between free and protein bound states. Binding of ¹³C-GalNAc was studied as a function of temperature. From the temperature dependence of the line broadening, the thermodynamic and kinetic parameters were evaluated. The association rate constants obtained for the α-anomers of ¹³C-GalNAc and ¹⁹F-GalNAc (k₊₁=1.01×10⁵ M⁻¹•s⁻¹ and 0.698 ×10⁵ M⁻¹•s⁻¹, respectively) are in close agreement with those obtained for the corresponding β-anomers (k₊₁=0.95×10⁵ M⁻¹•s⁻¹ and 0.65×10⁵ M⁻¹•s⁻¹, respectively), suggesting that the two anomers bind to the lectin by a similar mechanism. In addition these values are several orders of magnitude slower than those obtained for diffusion controlled processes. The dissociation rate constants obtained are 49.9, 56.9, 42, and 43 s⁻¹, respectively, for the α- and β-anomers of ¹³C-GalNAc and ¹⁹F-GalNAc. A two-step mechanism has been proposed for the interaction of ¹³C-GalNAc and ¹⁹F-GalNAc with A. integrifolia lectin in view of the slow association rates and high activation entropies. The thermodynamic parameters obtained for the association and dissociation reactions suggest that the binding process is entropically favored and that there is a small enthalpic contribution.