Binding interactions of acrylamide with lysozyme and its underlying mechanisms based on multi-spectra, isothermal titration microcalorimetry and docking simulation

Abstract

Acrylamide is a known carcinogenic and neurotoxic organic compound that is widespread in the environment and poses potential threats to human beings and other living organisms. Herein, we explored the interaction mechanism of acrylamide with lysozyme via multiple spectroscopic approaches, ITC (isothermal titration calorimetry), enzyme activity, as well as molecular docking under physiological conditions. Results suggested that acrylamide can interact with lysozyme fluorophore residues including Trp and Tyr, and exhibit a fluorescence sensitization ability toward these amino acids. The direct interaction of lysozyme with acrylamide resulted in the unfolding of polypeptide chains along with the loosening of protein skeletons, decreasing the α-helical content, and hence a smaller size of lysozyme agglomerated. Moreover, acrylamide docked to lysozyme at relatively low affinity and the interactions were primarily driven by van der Waals and hydrogen bonding interactions. Further molecular analyses illustrated that the binding site was around the active center of lysozyme, acrylamide interacted with Glu 35, Asn 44, Asn 46, Asp 52, Gln 57, Ala 107, Trp 108 and Val 109. The molecular lysozyme activity was repressed due to the direct cross talk of acrylamid with the key residues Glu 35, as well as Asp 52 of the enzyme activity near the active center lysozyme. In addition, the fluorophore residues Trp 108 was also in the binding pocket, which can also explain the fluorescence sensitization phenomenon. Taken together, our study demonstrated the structure and function of lysozyme were both affected by acrylamide.