Serum Prolidase in Myocardial Infarction: Purification, Kinetic Properties, and Drug-Enzyme Interactions

Abstract

Objective: This study aimed to isolate, purify, and characterize the enzyme prolidase from the serum of patients with myocardial infarction (MI). Also, evaluate potential pharmaceutical inhibitors using in silico methods.Methods: Prolidase was purified using a multi-step biotechnological protocol comprising ammonium sulphate precipitation, dialysis, affinity chromatography (Sepharose 4B-procyanidin), and gel filtration (Sephadex G-100). The physicochemical properties and kinetic parameters of the purified enzyme were determined. Additionally, molecular docking studies were conducted to assess the binding interactions of five pharmaceutical compounds-aspirin, nitroglycerin, propranolol, empagliflozin, and losartan-against the prolidase active site (PDB ID: 2OKN).Results:The purification protocol yielded a specific activity of 1354.9 U/mg protein with a purification fold of 40.12. The molecular weight of the isolated enzyme was determined to be approximately 51000±250 Daltons via electrophoresis. Optimal enzyme activity was observed in 65 mM Tris-HCl buffer at pH 8.0 and 40°C, with a substrate (Gly-Pro) concentration of 15.0 mM. Kinetic characterization using Lineweaver-Burk plots revealed a Michaelis-Menten constant (K m ) of 23.42 mM and a maximum velocity (V max ) of 40.32 U/mL. Molecular docking analysis indicated that among the tested compounds, losartan and empagliflozin exhibited the strongest binding affinities, with histidine-378 (His378) identified as a key residue in the enzyme-inhibitor interaction.Conclusion:The study successfully characterized prolidase from MI patients and highlighted the potential of losartan and empagliflozin as interacting ligands, suggesting avenues for further pharmacological investigation.