Controllable enzymatic superactivity of α-chymotrypsin activated by the electrostatic interaction with cationic gemini surfactants

Surfactant plays a critical role in enzymatic multi-functionalization processes. However, a deep understanding of surfactant-enzyme interactions has been lacking up until now due to the extreme complexity of the mixed system. This work reported the effect of cationic gemini surfactants, alkanediyl-alpha,omega-bis(dimethyldodecylammonium bromide) (C12CSC12Br2, S = 2, 6, and 10) on the enzymatic activity and conformation of alpha-chymotrypsin (alpha-CT) in phosphate buffer solution (PBS, pH 7.3). The enzymatic activity was assessed by the rate of 2-naphthyl acetate (2-NA) hydrolysis measured by UV-vis absorption. The superactivity of alpha-CT in the presence of C12CSC12Br2 appears in the concentration region below the critical micelle concentration (cmc) of the surfactant, and its maximum superactivity is correlated to the spacer length of C12CSC12Br2. Subtle regulation of the charge density of headgroups of the cationic surfactant can be achieved through partial charge neutralization of cationic headgroups by introducing inorganic counterions or oppositely charged surfactant, demonstrating that the electrostatic interaction plays the crucial role for emergence of the superactivity. The interaction between C12CSC12Br2 (S = 2,6, and 10) and alpha-CT was characterized by isothermal titration calorimetry (ITC), and the obtained endothermic enthalpy change indicates that the interaction induces the change in conformation and enzymatic superactivity. The methodologies of fluorescence spectroscopy, circular dichroism (CD), and differential scanning calorimetry (DSC) show that the gemini surfactants with different spacer lengths induct and regulate the secondary, tertiary and even fourth structures of the protein. The present work is significant to get deeper insight into the mechanism of the activation and denaturation of enzymes.

Automated summary

This study investigated the impact of cationic gemini surfactants on the enzymatic activity and conformation of alpha-chymotrypsin (α-CT), finding that the surfactants led to superactivity of α-CT below the critical micelle concentration, and the charge density of headgroups was regulated through electrostatic interactions. Characterization using isothermal titration calorimetry (ITC) revealed an endothermic enthalpy change, indicating a change in conformation and enzymatic superactivity. Fluorescence spectroscopy, circular dichroism (CD), and differential scanning calorimetry (DSC) demonstrated that the gemini surfactants induced and regulated different protein structures. The findings are significant for understanding the activation and denaturation mechanisms of enzymes.