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<p>Corn protein was hydrolyzed by alkaline protease and separated into three fractions (&lt; 1, 1–3, &gt; 3&#xa0;kDa) through ultrafiltration. The fraction &lt; 1&#xa0;kDa exhibited a better inhibitory effect on α-amylase in the three fractions, and it was further purified into nine fractions (F1–F9) by reverse-phase high-performance liquid chromatography (RP-HPLC). Three peptides (WGPQ, FPGPK, PGFK) were identified and screened from F3 and synthesized. WGPQ, FPGPK, and PGFK demonstrated a great α-amylase inhibition with semi-inhibitory concentrations (IC<sub>50</sub>) of 915.12&#xa0;μg/mL, 790.59&#xa0;μg/mL, and 1020.77&#xa0;μg/mL, respectively. The inhibitory effect of corn peptides on α-amylase was mixed (competitive and non-competitive) inhibition. The peptide sequence containing Gly, Pro, and Phe was an important feature of α-amylase inhibition. Molecular docking analysis indicated that three peptides affected the flexible loop or conformational rigidity of α-amylase through hydrogen bonds and hydrophobic interactions. Pi-Pi stacks and salt bridges were also formed between peptides and specific amino acids of α-amylase. Isothermal titration calorimetry results indicated that molecular binding was more significantly influenced by hydrophobic forces and hydrogen bonds. The results of circular dichroic chromatography showed that the forces that maintain the secondary structure of amylase were changed, especially under the action of peptides containing Gly and Pro. It indicated that corn peptides spontaneously bond to the active site of α-amylase and changed its structure. This study provided the theoretical basis and effective strategy for the application of active peptides in diabetes.</p>

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Mechanistic Inhibition of α-Amylase by Corn Peptide: Site-Specific Binding Induces Conformational Destabilization and Enzymatic Activity Suppression

  • 期刊论文

  • 发表日期：

    2025年6月30日

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  • Shu Wang,
  • Song Zhu,
  • Dejian Huang,
  • Yue Li

摘要

Corn protein was hydrolyzed by alkaline protease and separated into three fractions (< 1, 1–3, > 3 kDa) through ultrafiltration. The fraction < 1 kDa exhibited a better inhibitory effect on α-amylase in the three fractions, and it was further purified into nine fractions (F1–F9) by reverse-phase high-performance liquid chromatography (RP-HPLC). Three peptides (WGPQ, FPGPK, PGFK) were identified and screened from F3 and synthesized. WGPQ, FPGPK, and PGFK demonstrated a great α-amylase inhibition with semi-inhibitory concentrations (IC50) of 915.12 μg/mL, 790.59 μg/mL, and 1020.77 μg/mL, respectively. The inhibitory effect of corn peptides on α-amylase was mixed (competitive and non-competitive) inhibition. The peptide sequence containing Gly, Pro, and Phe was an important feature of α-amylase inhibition. Molecular docking analysis indicated that three peptides affected the flexible loop or conformational rigidity of α-amylase through hydrogen bonds and hydrophobic interactions. Pi-Pi stacks and salt bridges were also formed between peptides and specific amino acids of α-amylase. Isothermal titration calorimetry results indicated that molecular binding was more significantly influenced by hydrophobic forces and hydrogen bonds. The results of circular dichroic chromatography showed that the forces that maintain the secondary structure of amylase were changed, especially under the action of peptides containing Gly and Pro. It indicated that corn peptides spontaneously bond to the active site of α-amylase and changed its structure. This study provided the theoretical basis and effective strategy for the application of active peptides in diabetes.