Ishikawa K, Hirata H
National Chemical Laboratory for Industry, Ibaraki, Japan.
Arch Biochem Biophys. 1989 Aug 1;272(2):356-63. doi: 10.1016/0003-9861(89)90229-4.
Conversion of the substrate specificity of porcine pancreatic alpha-amylase (PPA) was studied using chemical modification of His residues. Diethyl pyrocarbonate modified His residues in PPA and the activity of the modified PPA for the hydrolysis of the alpha-D-(1,4)glucoside bond in starch or oligosaccharides decreased to less than 1% of that of the native enzyme. However, the activity for the hydrolysis of the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides was increased by chemical modification. When the modified PPA was incubated with a proteinaceous alpha-amylase inhibitor (Mr 60,000) purified from white kidney bean (Phaseolus vulgaris), it bound to the inhibitor. As a result, the remaining less than 1% hydrolytic activity of the modified PPA for starch disappeared completely but that for p-nitrophenyl oligosaccharides remained unaltered. The hydrolytic activity of the native PPA for the alpha-D-(1,4)glucoside bond in oligosaccharides was stronger than that between p-nitrophenyl and oligosaccharides in p-nitrophenyl oligosaccharides. Therefore, when p-nitrophenyl oligosaccharides (three to five glucose residues) were used as substrates for the native PPA, the alpha-D-(1,4)glucoside bonds in the oligosaccharides were hydrolyzed. However, the modified PPA-inhibitor complex hydrolyzed only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides. The above results reveal that, by chemical modification with diethyl pyrocarbonate and biochemical modification with an amylase inhibitor, amylase can be converted to a new exo-type enzyme which hydrolyzes only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides.
利用组氨酸残基的化学修饰研究了猪胰α-淀粉酶（PPA）底物特异性的转变。焦碳酸二乙酯修饰了PPA中的组氨酸残基，修饰后的PPA对淀粉或寡糖中α-D-(1,4)糖苷键的水解活性降至天然酶的1%以下。然而，化学修饰增加了其对对硝基苯基寡糖中对硝基苯酚与寡糖之间键的水解活性。当将修饰后的PPA与从白芸豆（菜豆属）中纯化的一种蛋白质类α-淀粉酶抑制剂（分子量60,000）一起孵育时，它与该抑制剂结合。结果，修饰后的PPA对淀粉剩余的不到1%的水解活性完全消失，但对硝基苯基寡糖的水解活性保持不变。天然PPA对寡糖中α-D-(1,4)糖苷键的水解活性强于对硝基苯基寡糖中对硝基苯基与寡糖之间的水解活性。因此，当以对硝基苯基寡糖（三到五个葡萄糖残基）作为天然PPA的底物时，寡糖中的α-D-(1,4)糖苷键被水解。然而，修饰后的PPA-抑制剂复合物仅水解对硝基苯基寡糖中对硝基苯酚与寡糖之间的键。上述结果表明，通过焦碳酸二乙酯的化学修饰和淀粉酶抑制剂的生化修饰，淀粉酶可以转化为一种新的外切型酶，该酶仅水解对硝基苯基寡糖中对硝基苯酚与寡糖之间的键。
