Pb^(2+)对α-淀粉酶活性的影响及其光谱学研究

Study of the Effect of Pb ^(2+) on α-Amylase Activity by Spectroscopy

在α 淀粉酶介质中加入Pb2 + ,通过光谱学手段研究Pb2 + 对α 淀粉酶活性影响的作用机理。结果表明低浓度的Pb2 + 对酶有激活作用 ,高浓度则严重抑制酶活性。在高浓度下 ,Pb2 + 能完全竞争出α 淀粉酶中的Ca2 + 而结合到了α 淀粉酶上 ,其EXAFS的测试表明Pb2 + 与氨基酸残基上的羧基氧发生了配位 ,配位数为 2 ,Pb—O键长为 0 2 34nm。圆二色 (CD)谱测试表明 ,高浓度的Pb2 + 结合使α 淀粉酶的二级结构被破坏 ,α 螺旋含量、β 转角及无规则卷曲大量下降 ,β 折叠、二硫键含量大量增多 ,Pb2 + 的这种完全结合致使酶的构象改变 ,形成无效的酶 Pb2 + 底物复合物 ,因而使酶失去活性。

The activity of α-amylase from porcine pancreas was enhanced under the treatment by Pb 2+ at low concentration (0.5-4 μmol·L -1),but was inhibited by Pb 2+ at high concentration (above 4 μmol·L -1).Pb 2+ at high concentration could competitively displace Ca 2+ from α-amylase. The EXAFS demonstrated that Pb 2+ was bound to the active site of α-amylase, the coordination atom was oxygen,the coordination number was 2, and the Pb-O bond length was 0.234 nm.Circular dichroism spectra showed that the secondary structure of trypsin was greatly changed by Pb 2+ at high concentration,as α-helix,β-turn and random coil contents decreased,while β-sheet, aromatic and disulfide bond contents increased. It was suggested that Pb 2+ was bound to result in an α-amylase conformational change, and the enzyme activity decreased.