血管紧张素转化酶抑制肽Leu-Lys-Pro(LKP)抑制机理的研究

血管紧张素转化酶(ACE)是一种含锌离子的羧二肽酶，通过肾素-血管紧张素系统和激肽释放酶-激肽系统进行血压调节。食源血管紧张素转化酶抑制肽(ACEIP)可抑制ACE的活性对高血压控制有利。以鲣鱼蛋白分离出的ACE抑制肽Leu-Lys-Pro(LKP)为原料，采用荧光光谱法、紫外-可见光谱法、圆二色谱法、等温滴定量热法(ITC)以及分子对接技术研究了LKP对ACE的抑制机理。荧光光谱结果表明，LKP能够有效猝灭ACE的内源荧光，猝灭机制为静态猝灭，两者结合可形成较稳定的复合物，ACE中色氨酸和酪氨酸残基所处的微环境疏水性减小，导致极性增强。紫外、圆二色谱结果表明，LKP与ACE结合会导致ACE构象发生改变，ACE与LKP结合后二级结构比未结合时松散，为紧密-松散-稍紧密的变化过程。ITC测得LKP与ACE结合的焓变(ΔH)、熵变(ΔS)、化学计量比(n)以及结合常数(K_a)等热力学参数，结果表明两者结合反应是由熵驱动的自发吸热过程，结合力主要为疏水作用，确定LKP与ACE相互作用的结合位点数约为1,且随温度升高而增加。LKP与ACE的结合常数K_a

Study on the Inhibition Mechanism of Angiotensin Conversion Enzyme Inhibitor Peptide Leu-Lys-Pro (LKP)

Angiotensin-Ⅰ Converting Enzyme (ACE) is a zinc-containing carboxydipeptidase that regulates blood pressure through renin-angiotensin and kallikrein-kinin systems. The ACE inhibitory peptide (ACEIP) derived from food protein could inhibit the activity of ACE, which is beneficial to antihypertension. In this paper, the inhibition mechanism of the inhibitory peptide LKP from bonito fish on ACE was studied by using fluorescence spectra, ultraviolet absorption spectra, circular dichroism (CD), and isothermal titration calorimetry (ITC) and molecular docking. The fluorescence spectra showed that LKP could effectively quench the endogenous fluorescence of ACE, and the quenching mechanism was static quenching by the formation of a relatively stable complex LKP-ACE. The microenvironment around the tryptophan and tyrosine residues in ACE was localized, decreased the hydrophobicity, and enhanced the polarity. The results of UV and CD showed that the combination of LKP and ACE would lead to the conformation change of ACE. After the addition of LKP, the secondary structure of ACE became looser, and the structural changes of a tightness, loosening and slightly tighter have taken place during the interaction process. The thermodynamic parameters such as enthalpy change (ΔH), entropy change (ΔS), stoichiometric ratio (n) and binding constant (K_a) of the interaction between LKP and ACE were obtained by the ITC method. The results showed that the binding reaction of LKP and ACE was a spontaneous endothermic process driven by entropy, and the binding force was mainly hydrophobic. The stoichiometric ratio (n) value was determined to be about 1, which was enhanced with increasing temperature. At 288, 293 and 299 K, the binding constants Ka of LKP and ACE were 2.2×103, 0.9×103 and 5.3×103, respectively, indicating the affinity of LKP and ACE was relatively low. The results of molecular docking showed that the amino acid residues Gln281 and Lys511 in the S₁ pocket of the ACE active center could form two hydrogen bonds with LKP, and hydrophobic interaction could have occurred between His353 and His513 and LKP, LKP bond to ACE mainly through hydrophobicity, and hydrogen bonds stabilized the spatial structure of the protein. This study provides certain help for exploring the interaction between ACE inhibitory peptide and ACE and offers some theoretical basis for the development of new hypertension drugs.