菜青虫N-乙酰-β-D-氨基葡萄糖苷酶活性必需基团的研究

Studies on the Essential Groups of the β-N-acetyl-D-glucosaminidase from the Cabbage Butterfly(Pieris brassicae)

从菜青虫表皮分离纯化N-乙酰-β-D-氨基葡萄糖苷酶(NAGase),获得电泳单一纯的酶制剂,通过化学修饰法研究该酶的活性必需基团.分别以碳化二亚胺(EDC)、N-溴代琥珀酰亚胺(NBS)、对-氯汞苯甲酸(pCMB)、二硫苏糖醇(DTT)、溴代乙酸(BrAc)和乙酰丙酮在特定条件下对该酶进行特异的化学修饰,结果表明酸性氨基酸的侧链羧基、半胱氨酸巯基、色氨酸的吲哚基、组氨酸的咪唑基、二硫键被修饰后,酶活性均显著下降,因此这些氨基酸残基是酶活性的必需基团,而精氨酸残基及赖氨酸ε-氨基被修饰后对酶活力没有影响,不是酶的必需基团.

A β-N-acetyl-D-glucosaminidase（NAGase） was purified from the integument of the larva of cabbage butterfly（Pieris brassicae）. The purified enzyme was determined to be homogeneous by polyacrylamide gel electrophoresis（PAGE）. The characters of functional groups of the enzyme active site have been studied using chemical modification method. The enzyme was modified respectively by several chemical modification reagents, such as carbidiimide（EDC）, pCMB（p-chloromercuribenzoate）, N-bromosuccinimide （NBS）, bromoacetic acid（BrAe）, DTT（dithiothreitol）, acetic anhydride and acetyl acetone at certain condition, and the residue activity was assayed in normal reaction media. The results showed that the enzyme activity was decreased when the carboxyl group, sulfhydryl group,indolyl group,imidazolyl group and disulfide bond were modified, respectively, which means that these groups were essential for the enzyme catalytic activity. The results also showed that the residues of lysine, arginine were irrespective with the enzyme activity.