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

Area of Science:

• Biochemistry
• Enzymology
• Protein Structure and Function

Background:

• Carboxypeptidase A (CPA) is a key metalloproteinase in the M14 family, crucial for cleaving C-terminal amino acids from polypeptides.
• Understanding CPA's interactions with small molecules is vital for elucidating its catalytic mechanisms and potential therapeutic applications.

Purpose of the Study:

• To investigate the impact of spermidine on the conformation, thermal stability, and enzymatic activity of native bovine pancreatic CPA.
• To characterize the binding interactions between CPA and spermidine at a molecular level.

Main Methods:

• Utilized UV-Vis spectroscopy, intrinsic fluorescence, circular dichroism (CD), and thermal stability assays (Tm studies).
• Employed kinetic techniques to assess enzyme activity and molecular docking for interaction analysis.

Main Results:

• Spermidine formed complexes with CPA, indicated by decreased UV-Vis absorption and static fluorescence quenching.
• Binding was spontaneous, stabilized by van der Waals and hydrogen interactions, with spermidine partially altering CPA's secondary structure.
• Spermidine increased CPA's thermal stability (Tm) and enhanced its catalytic activity (Vmax).

Conclusions:

• Spermidine positively modulates bovine CPA's properties, enhancing its stability and enzymatic efficiency.
• The findings provide insights into CPA-ligand interactions and the role of polyamines in enzyme modulation.