Asen Nancy D, Okagu Ogadimma D, Udenigwe Chibuike C, Aluko Rotimi E
Department of Food and Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, Canada.
Department of Chemistry and Biomolecular Sciences, Faculty of Science, University of Ottawa, Ottawa, ON, Canada.
Front Nutr. 2022 Oct 21;9:1021893. doi: 10.3389/fnut.2022.1021893. eCollection 2022.
Compounds with structural similarities to the neurotransmitter (acetylcholine) are mostly used to inhibit the activity of acetylcholinesterase (AChE) in Alzheimer's disease (AD) therapy. However, the existing drugs only alleviate symptoms of moderate to mild conditions and come with side effects; hence, the search is still on for potent and safer options. In this study, High performance liquid chromatography (HPLC) fractionations of AChE-inhibitory pea protein hydrolysates obtained from alcalase, flavourzyme and pepsin digestions were carried out followed by sequence identification of the most active fractions using mass spectrometry. Subsequently, 20 novel peptide sequences identified from the active fractions were synthesized and five peptides, QSQS, LQHNA, SQSRS, ETRSQ, PQDER (IC = 1.53 - 1.61 μg/mL) were selected and analyzed for ability to change AChE protein conformation (fluorescence emission and circular dichroism), kinetics of enzyme inhibition, and enzyme-ligand binding configurations using molecular docking. The kinetics studies revealed different inhibition modes by the peptides with relatively low (<0.02 mM and <0.1 mM) inhibition constant and Michaelis constant, respectively, while maximum velocity was reduced. Conformational changes were confirmed by losses in fluorescence intensity and reduced α-helix content of AChE after interactions with different peptides. Molecular docking revealed binding of the peptides to both the catalytic anionic site and the peripheral anionic site. The five analyzed peptides all contained glutamine (Q) but sequences with Q in the penultimate N-terminal position (LQHNA, SQSRS, and PQDER) had stronger binding affinity. Results from the different analysis in this study confirm that the peptides obtained from enzymatic digestion of pea protein possess the potential to be used as novel AChE-inhibitory agents in AD management.
在阿尔茨海默病（AD）治疗中，与神经递质（乙酰胆碱）结构相似的化合物大多用于抑制乙酰胆碱酯酶（AChE）的活性。然而，现有药物仅能缓解轻至中度症状且伴有副作用；因此，人们仍在寻找更有效且更安全的选择。在本研究中，对通过碱性蛋白酶、风味酶和胃蛋白酶消化获得的具有AChE抑制活性的豌豆蛋白水解物进行了高效液相色谱（HPLC）分级分离，随后使用质谱对活性最高的级分进行序列鉴定。随后，合成了从活性级分中鉴定出的20个新的肽序列，并选择了5个肽QSQS、LQHNA、SQSRS、ETRSQ、PQDER（IC = 1.53 - 1.61 μg/mL），并利用分子对接分析它们改变AChE蛋白构象（荧光发射和圆二色性）、酶抑制动力学以及酶 - 配体结合构型的能力。动力学研究表明，这些肽具有不同的抑制模式，其抑制常数和米氏常数相对较低（分别<0.02 mM和<0.1 mM），同时最大速度降低。与不同肽相互作用后，AChE的荧光强度损失和α - 螺旋含量降低证实了构象变化。分子对接显示这些肽与催化阴离子位点和外周阴离子位点均有结合。所分析的5个肽均含有谷氨酰胺（Q），但倒数第二个N端位置含有Q的序列（LQHNA、SQSRS和PQDER）具有更强的结合亲和力。本研究中不同分析的结果证实，豌豆蛋白酶解得到的肽具有在AD管理中用作新型AChE抑制剂的潜力。
