Shi Yan, Jiang Zhe, Han Peng, Zheng Guo-Xing, Song Kang-Kang, Chen Qing-Xi
Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, People's Republic of China.
Biochimie. 2007 Mar;89(3):347-54. doi: 10.1016/j.biochi.2006.06.016. Epub 2006 Aug 4.
A beta-N-acetyl-D-glucosaminidase (NAGase) from the cabbage butterfly (Pieris rapae) was purified. The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific activity was determined to be 8715 U/mg. The molecular weight of whole enzyme was determined to be 106 kDa by gel filtration, and the result of SDS-PAGE showed that the enzyme was a heterodimer, which contained two subunits with different mass of 59.5 and 57.2 kDa. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) were investigated to be at pH 6.2 and at 42 degrees C, respectively, and the Michaelis-Menten constant (K(m)) was determined to be 0.285 mM at pH 6.2 and 37 degrees C. The stability of the enzyme was investigated and the results showed that the enzyme was stable at the pH range from 4.0 to 9.0 and at the temperature below 45 degrees C. The activation energy was 83.86 kJ/mol. The reaction of this enzyme with pNP-NAG was judged to be Ordered Bi-Bi mechanism according to the inhibitory behaviors of the products. The ionization constant, pK(e), of ionizing group at the active site of the enzyme was found to be 5.20 at 39.0 degrees C, and the standard dissociation enthalpy (DeltaH(o)) was determined to be 2.18 kcal/mol. These results showed that the ionizing group of the enzyme active center was the carboxyl group. The results of chemical modification also suggested that carboxyl group was essential to the enzyme activity. Moreover, Zn(2+), Hg(2+), Cu(2+) had strongly inhibitory effects on the enzyme activity.
从菜粉蝶（粉纹夜蛾）中纯化出一种β-N-乙酰-D-氨基葡萄糖苷酶（NAGase）。纯化后的酶在聚丙烯酰胺凝胶电泳上呈现单一条带，比活性测定为8715 U/mg。通过凝胶过滤法测定全酶的分子量为106 kDa，SDS-PAGE结果表明该酶是一种异二聚体，由质量分别为59.5 kDa和57.2 kDa的两个亚基组成。研究了该酶水解对硝基苯基-N-乙酰-β-D-氨基葡萄糖苷（pNP-NAG）的最适pH和最适温度，分别为pH 6.2和42℃，在pH 6.2和37℃下测定米氏常数（K(m)）为0.285 mM。对该酶的稳定性进行了研究，结果表明该酶在pH 4.0至9.0范围内以及温度低于45℃时稳定。活化能为83.86 kJ/mol。根据产物的抑制行为判断该酶与pNP-NAG的反应为有序双底物双产物机制。在39.0℃下发现该酶活性位点的电离基团的电离常数pK(e)为5.20，标准解离焓（ΔH(o)）测定为2.18 kcal/mol。这些结果表明该酶活性中心的电离基团是羧基。化学修饰结果也表明羧基对酶活性至关重要。此外，Zn(2+)、Hg(2+)、Cu(2+)对酶活性有强烈抑制作用。
