大黄酸对α-淀粉酶的抑制机理分析及分子模拟

Inhibitory Mechanism of Rhein on α-Amylase and Its Molecular Simulation

摘要:

本文应用紫外、荧光、圆二色谱和分子模拟等手段，分析测定了大黄酸对α-淀粉酶的抑制作用、抑制类型和抑制动力学常数，探讨了大黄酸对α-淀粉酶二级结构的影响，并通过分子模拟软件对大黄酸和α-淀粉酶进行分子对接。结果表明，大黄酸是一种可逆的竞争型α-淀粉酶抑制剂，其半抑制率IC50为0.43±0.02 mM，抑制常数Ki为0.35±0.03 mM；进一步的圆二色谱分析表明，大黄酸能使α-淀粉酶二级结构含量的变化（α-螺旋含量由12.22%逐渐增加到22.72%，β-折叠含量由43.24%逐渐降低到36.43%），这意味着大黄酸能够诱导α-淀粉酶的构象发生部分改变；螯合钙离子实验证实了大黄酸能够螯合活性中心的钙离子；分子模拟结果也表明，大黄酸能够优先结合到α-淀粉酶的活性中心，并与催化基团TRP59和TYR62发生相互作用，形成氢键和π?π效应，进而与淀粉竞争活性中心，从而降低了α-淀粉酶的催化活性。

Abstract:

The inhibitory effect (including inhibitory mechanism, type and kinetic parameters) of rhein on α-amylase and the effect on α-amylase secondary structure were investigated by UV, fluorescence, and circular dichroism spectrometry, while molecular docking of rhein and α-amylase was conducted by molecular simulation. The results showed that rhein was a reversible and competitive inhibitor to α-amylase. The half-maximal inhibitory concentration (IC50) and inhibition constant (Ki) were 0.43 ± 0.02 mM and 0.35 ± 0.03 mM, respectively. Circular dichroism spectra analysis revealed that rhein could alter the content of secondary structure in α-amylase (gradual increase of α-helix content from 12.22% to 22.72% and gradual decrease of β-sheet from 43.24% to 36.43%), indicating that rhein could induce partial conformational changes in α-amylase. Chelating calcium ions experiment indicated that rhein combined with calcium ions at the α-amylase active site. Moreover, the results of molecular simulation indicated that rhein had a binding preference for the α-amylase active site, interacting with the catalytic groups TRP59 and TYR62 by forming hydrogen bonds and π-π interaction. Furthermore, rhein competed with starch, consequently reducing the catalytic activity of α-amylase.