Study On The Inhibition Mechanism Of Angiotensin Conversion Enzyme With Inhibitor Peptide Leu-Lys-Pro(LKP)

Active peptides extracted from natural protein sources can affect a variety of physiological processes.Among them,peptides that inhibit the activity of Angiotensin-ⅠConverting Enzyme（ACE）are called angiotensin-inhibiting peptides,which are safe,mild and less side effects.It could play an auxiliary role in the treatment of hypertension.The inhibition mechanism of the binding of inhibitor peptides to ACE is of great significance for the development and application of ACE inhibitor peptides.In this study,Leu-Lys-Pro（LKP）,a tripeptide extracted from bonito protein,was selected as the research object to explore the mechanism of LKP’s inhibition to ACE in HEPES and ACES buffer fluid system,mainly including the following research contents:ACE crude enzyme solution was obtained from fresh pig lung after several purification steps including homogenization,ammonium sulfate fractionation salting out and dialysis.The crude enzyme solution was also purified by DEAE-FF anion exchange chromatography-ultrafiltration method,to obtain the intercepted solution.After a series of separations and purification,the electrophoretic purity of ACE was obtained.The specific activity of ACE reached 1.87 U/mg,with the total enzyme activity recovery35.9%,and the purification ratio was 311.7.The inhibitory types and IC₅₀ values of LKP were investigated.Inhibition type of LKP was determined to be mixed type by Lineweaver-Buck Double reciprocal plotting method.The IC₅₀ value of inhibitory peptide LKP was 0.318μmol/L.Thermodynamics of LKP’s inhibition of ACE was investigated,thermodynamic parameters of the combination of LKP and ACE were measured by Isothermal titration calorimetry（ITC）experiments:enthalpy change（ΔH）,entropy change（ΔS）,stoichiometric ratio（n）and binding constant（K_a）,which could judge the non-covalent bonding force of the binding reaction.The experimental results showed that the binding reaction between LKP and ACE was spontaneous exothermal reaction.The binding force in HEPES and ACES buffer is mainly entropy driven,and the non-covalent bonding force is mainly hydrophobic.LKP mainly inhibits ACE activity by changing the spatial conformation of ACE through hydrophobic interaction between LKP and ACE amino acid residues.The effect of LKP on the conformation of ACE space was further studied by ultraviolet absorption spectroscopy,fluorescence spectroscopy and circular dichroism（CD）techniques.The experimental results show that LKP could bind to ACE to form a new stable complex,quenching the endogenous fluorescence of ACE with a static quenching mechanism.After the binding of LKP to ACE reaches saturation,the spatial structure of ACE still changed,indicating that although the free LKP is no longer bound to ACE,it can still affect the spatial conformation of the ACE.Results of CD spectra showed that the secondary structure of ACE was changed by binding to LKP,and the protein structure was loosened first and then folded tightly.The final structure of ACE in HEPES buffer was looser than without LKP added,and the final structure of ACE in ACES buffer was tighter than without LKP.The binding model of LKP and ACE was studied by molecular docking technology.The results showed that in HEPES system,the optimal binding conformation was LKP binding at ACE active site,while in ACES system,the optimal binding conformation was LKP binding at ACE inactive site.Both LKP and ACE amino acid residues were bound by hydrophobic interaction and hydrogen bonding.