Comparative studies on the interaction of spermidine with carboxypeptidase A using multispectroscopic and docking methods

Abstract

Carboxypeptidase A (CPA) (EC 3.4.17.1) is one of the main members of the M14 family that release one amino acid from the C-terminal region of the polypeptides at each time. The purpose of the present study was to study the effect of spermidine (NH2(CH2)3NH(CH2)4NH2) on the conformation, thermal stability, and activity of native CPA from bovine pancreas, by employing ultraviolet–visible (UV–Vis) spectroscopy, intrinsic fluorescence, thermal stability, circular dichroism (CD), kinetic techniques and molecular docking. It was found that the decrease in the CPA, UV–Vis absorption could be due to the formation of the CPA–spermidine complexes. The results of fluorescence spectroscopic measurements at the temperatures of 308 and 318 K also revealed that spermidine had the capability to quench the intrinsic fluorescence of CPA with the static mode. Further, the thermodynamic parameters, (Gibbs free-energy, enthalpy and entropy changes) showed that the binding process of spermidine to CPA was spontaneous and the main force in stabilizing the complex was the van der Waals and hydrogen interactions, along with the molecular docking results. In addition, CD spectra and fluorescence results revealed that spermidine had a partial effect on the CPA structure, leading to some changes in its secondary structure. The Tm studies of the CPA–spermidine complex also indicated that the Tm values were enhanced with increasing the spermidine concentration. Kinetic studies further showed that by spermidine binding, the Vmax value and activity of the enzyme were increased.