Research On The Domain Selective Inhibition Of Angiotensin Converting Enzyme (ACE) By Tyrosine-Containing Oligopeptides

Hypertension is one of the most common cardiovascular diseases and directly affects and harms people's health.Angiotensin converting enzyme(Angiotensin Converting Enzyme,ACE,EC 3.4.15.1)plays an important role in regulating blood pressure and controlling cardiovascular disease.ACE has two active domains,of which the C-domain is the major site of action for blood pressure regulation,and the N-domain has no significant effect on blood pressure regulation.The bioactive peptide ACE inhibitor peptides have attracted wide attention due to their small side effects and easy absorption.Therefore,it is necessary to clarify the mechanism of interaction between ACE two domains and bioactive small peptides,and then effectively inhibit ACE.In this paper,ACE inhibitor dipeptides containing tyrosine residues were investigated.The domain selective inhibitory activity,stability,interaction of ACE were studied.The main research contents and results are as follows:(1)Screening containing tyrosine dipeptide that selectively inhibits the C-domain of ACE.The results showed that the C-terminal was tyrosine and the N-terminus was an acidic,basic,aromatic,hydroxyl amino acid dipeptide,which was non-selective inhibition on the C domain of ACE;the N-terminal was an aliphatic amino acids dipeptide,and its inhibitory activity on the ACE C-domain was significantly higher than its inhibitory activity on the ACE N-domain.The effect of tyrosine residues at the terminal of ACE inhibitory peptide on ACE domain inhibitory activity was investigated.The results showed that the dipeptide Tyr-Ala(YA),Tyr-Leu(YL)and Tyr-Ile(YI)with N terminal of tyrosine residues had no significant difference inhibitory activity on the C-domain and N-domain of ACE.However,the dipeptides AY,LY and IY with tyrosine residues at the C-terminal have significant inhibitory effects on the selective inhibition of C-domain of ACE.The structure of tyrosine residues at the C terminal of ACE inhibitors is a favorable structure for inhibiting the C-domain of ACE.The inhibition type of dipeptides containing tyrosine residues at the C-terminal Ser-Tyr(SY),Ala-Tyr(AY),Leu-Tyr(LY),Arg-Tyr(RY),Ile-Tyr(IY),Phe-Tyr(FY),Glu-Tyr(EY),Val-Tyr(VY)were measured by high performance liquid chromatography(HPLC).The results showed that the dipeptides with N-terminal aliphatic amino acids IY and LY were competitively inhibited,and AY and VY were mixed inhibited,and the N-terminal was acidic,basic,aromatic,and hydroxyl amino acids,it was non-competitive.(2)Determination of the thermal stability and resistance to gastrointestinal tract digestion and absorption ability of AY,IY,LY and SY,which contain tyrosine residue ACE inhibitor dipeptide,and optimize the quantitative analysis method of AY,IY,LY and SY.The results showed that the content of AY,IY,LY and SY has a good linear relationship in the concentration range of 0.01~0.02 mg/mL.The recovery rate was 98.41%~100.29%.The RSD of accuracy and precision were all less than 1%,and the detection limit was 2-3 ?g/mL.The detection method can determine the content of AY,IY,LY and SY in the sample.It is convenient and rapid and has high accuracy.IY,LY,AY and SY in 6 h at 37?,50? and 70? all have better thermal stability,and IY showed the best stability.AY,IY,LY and SY have good anti pepsin digestion and absorption capacity.After artificial gastric response for 2 h,the peptide loss rate was less than 10.00%.AY,IY,LY and SY have good anti-trypsin digestion and absorption capacity,and the peptide loss rate ranged from 12.73% to 16.01%,and the anti digestion and absorption ability of SY is lower than IY,LY and AY.Contain tyrosine residues ACE inhibitor dipeptide AY,IY,LY and SY with good thermal stability and good resistance to gastrointestinal tract digestion and absorption ability.(3)To study the conformation of ACE C-domain selective inhibitor IY and non-domain selective inhibitor SY in aqueous,dimethyl sulfoxide(DMSO)and methanol three solvents by nuclear magnetic resonance(NMR).Both in the aqueous solution were in a flexible and free extension conformation.In the methanol solution,they were in the extended state as the dominant conformation and accompanied by the folded conformation,while the DMSO solution were in the folded and collapsed state.The conformation changes of ACE after interaction of AY,IY,SY,LY and ACE were studied by circular dichroism,UV-visible spectroscopy and infrared spectroscopy.The four dipeptides all changed the conformation of ACE,which mainly changed the ?-helix and ?-fold of ACE.The conformational change of ACE with the selective inhibition of the C-domain and the competitive inhibition of IY was the most significant.The interaction between ACE domain and ACE inhibitor dipeptide with tyrosine residues was studied by molecular simulation.AY,IY,and LY have good affinity for the ACE C-domain,in which IY and LY have a high affinity for ACE C-domain.The N terminal amino acid of the dipeptide has a greater role in binding with the ACE C-domain than in the N-domain.The results of this study can provide a theoretical basis for the selective inhibition mechanism of ACE dipeptide with tyrosine residue.To provide a theoretical basis for studying the oral bioavailability of ACE inhibitory peptides containing tyrosine and specifically designing ACE C-domain selective inhibitory peptides.