Serum prolidase in myocardial infarction: purification, kinetic properties, and drug-enzyme interactions

Abstract

This study focused on the isolation, purification, and biochemical characterization of the prolidase from the serum of patients with myocardial infarction (MI), alongside an in silico evaluation of potential pharmaceutical inhibitors. Purification was achieved through a systematic biotechnological framework involving ammonium sulphate precipitation, dialysis, Sepharose 4B-procyanidin affinity chromatography, and Sephadex G-100 gel filtration, yielding a specific activity of 1354.9 U mg⁻¹ protein and a 40.12-fold purification. The isolated enzyme exhibited a molecular weight of approximately 51 000 ± 250 Da, with an optimal catalytic activity recorded at 40 °C in a 65 mM Tris-HCl buffer at a pH of 8.0 and a 15.0 mM Gly-Pro substrate concentration. Kinetic analysis via Lineweaver–Burk plots determined a Michaelis–Menten constant (K_m) of 23.42 mM and a maximum velocity (V_max) of 40.32 U mL⁻¹. Furthermore, molecular docking simulations targeting the prolidase active site (PDB ID: 2OKN) revealed that among the tested compounds—aspirin, nitroglycerin, propranolol, empagliflozin, and losartan—losartan and empagliflozin possessed the highest binding affinities, with His378 identified as a critical residue for enzyme–ligand interaction. These findings provide a robust characterization of prolidase in the clinical context of MI and suggest that losartan and empagliflozin warrant further pharmacological investigation as potential modulating agents.