丙酮对锯缘青蟹N-乙酰-β-D-氨基葡萄糖苷酶活力与构象的影响  

Effect of Acetone on Activity and Conformation of NAGase from Green Crab(Scylla serrata)

作　　者：杨学敏[1] 王勤[1] 谢进金[1] 张继平[1] 林建成[1] 陈清西[1] 

机构地区：[1]厦门大学生命科学学院细胞生物学与肿瘤细胞工程教育部重点实验室,福建厦门361005

出　　处：《厦门大学学报（自然科学版）》2006年第2期261-263,共3页Journal of Xiamen University：Natural Science

基　　金：国家自然科学基金(40576066)资助

摘　　要：以丙酮为效应物,研究其对锯缘青蟹(Scylla serrata)N-乙酰--βD-氨基葡萄糖苷酶(NAGase)活力的影响,结果表明:该酶的剩余活力随着丙酮浓度增大而呈指数下降,当丙酮浓度达25%,酶的剩余活力仅为20%,说明丙酮对青蟹NA-Gase有明显的失活作用.导致酶活力丧失50%的丙酮浓度为7.5%.在较低丙酮浓度(<10%)的失活是可逆的反应过程.动力学研究表明,该酶的失活过程属于混合型,并进一步测定游离酶(E)和酶底物络合物(ES)与丙酮的结合常数(KI和KIS),分别为4.06%和10.49%,KI<KIS,说明底物存在对酶被丙酮的失活作用有一定的保护作用.应用荧光发射光谱研究青蟹NAGase经丙酮微扰后的分子构象变化情况,结果表明:丙酮对酶分子构象有显著的影响,酶的内源荧光强度随丙酮浓度增大而降低,说明酶分子中的生色基团Trp和Tyr残基的微环境发生了变化.The effect of acetone on the activity of N-acetyl-β-D-glucosaminidase （NAGase） from green crab （Scylla serrata） was investigated. The results showed that the remaining activity rapidly declined with increasing the concentrations of acetone. The acetone of concentration at 7, 5%0 could lead to 50% enzyme activity lost. The inactivation of acetone on the enzyme was reversible when the concentration was lower than 10 %, The kinetics showed that the inactivation of the enzyme in acetone solution was mix-typed. The combinatory constants of the free enzyme （KI） and the enzyme-substrate complex （KIS） were determined to be 4.06% and 10.49%, respectively. The value of KIS is larger than that of Kt,indicating a marked protective effect of the substrate on the inactivation of the enzyme. Conformational changes of the enzyme in different concentrations of acetone were measured by fluorescence spectra. The fluorescence emission peak intensity of the enzyme gradually decreased and the peak had slight red-shifted with increasing acetone concentration. The results suggested the microenvironment of tyrosine and tryptophan of enzyme has been affected.

关 键 词：锯缘青蟹 N-乙酰-Β-D-氨基葡萄糖苷酶 丙酮 失活作用 构象变化 

分 类 号：Q356.1[生物学—遗传学]