Paraoxonase 1 (PON1) is a calcium-dependent hydrolytic enzyme bound to high-density lipoproteins (HDL), whose activity and concentration are tightly linked to various oxidative stress-related pathologies, including cardiovascular and neurodegenerative diseases. Despite intensive research, no reliable method for direct concentration-based quantification of PON1 in biological fluids exists to date. Current approaches rely solely on kinetic activity measurements with non-physiological substrates or on inconsistent enzyme linked immunosorbent assays. The aim of this doctoral work was to establish fundamental research into Ca2+ ion exchange with lanthanide ions and the binding of aromatic ligands at the active site of PON1, which could be exploited for further enzyme quantification.
In this thesis, we systematically explained how Tb3+ ion binding affects the enzymatic activity of recombinant PON1 (rePON1) and, subsequently, its photophysical properties upon formation of a ternary complex with 2-hydroxyquinoline (2HQ). We demonstrated that Tb3+ ions reversibly inhibit the lactonase activity of rePON1 through a sequential two-step mechanism involving both the catalytic and structural binding sites. Kinetic modeling of dihydrocoumarin (DHC) hydrolysis progress curves confirmed an immediate, low-affinity exchange of the catalytic Ca2+ ion (KI1 = 0.48 μM), followed by a slower, high-affinity substitution at the structural site (KI2 = 0.91 nM). The inhibited enzyme can be reactivated by an excess of Ca2+ ions, indicating preservation of the overall protein fold during ion replacement.
In the central part of the work, we showed that 2HQ forms a photoluminescent ternary complex with rePON1 and Tb3+ ion, which exhibits characteristic Tb3+-dependent time-resolved phosphorescence with emission peaks at the expected wavelengths. The phosphorescence intensity correlated linearly with rePON1 concentration, suggesting the potential for independent concentration-based quantification of PON1.
The crystal structure of the rePON1:Tb3+:2HQ complex at 2.35 Å resolution (PDB 9R0Q) revealed partial substitution of catalytic Ca2+ ions with Tb3+ (47%), while the occupancy at the structural site was low (5%) under the tested conditions. The inhibitor 2HQ bound in the same manner as in the Ca2+-complex, without major structural deviations. Isothermal titration calorimetry (ITC) confirmed that 2HQ binds much weaker to the rePON1:Tb3+ complex compared to rePON1:Ca2+ (approximately 30-fold weaker). Similar results for the binding of 2HQ to the rePON1:Tb3+ complex were also obtained by titrating 2HQ while measuring the phosphorescence signal upon the formation of rePON1:Tb3+:2HQ complex.
Overall, this doctoral thesis laid structural and mechanistic foundations for the use of photoluminescent lanthanide complexes in PON1 quantification. Future studies should focus on the search and development of aromatic ligands with higher affinity for the rePON1:Tb3+ complex, since low-affinity ligands require high concentrations to fully occupy the PON1 active sites, potentially causing self-quenching and reducing signal intensity and reliability. Therefore, we additionally performed non-targeted screening of ligands from a library of 1,409 drugs approved by the Chinese National Medical Products Administration, leading to the identification of prulifloxacin and efavirenz as promising candidates with 2- do 3-times greater binding affinities for rePON1 compared to 2HQ. These findings form an important starting point for future development of a system capable of detecting endogenous PON1 in complex biological samples.
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