锯缘青蟹NAGase在DMSO溶液中的失活动力学

Inactivation kinetics of N-acetyl-β-D-glucosaminidase from Scylla serrata by dimethylsulfoxide

以二甲亚砜(DMSO)为效应物,研究其对锯缘青蟹N-乙酰-β-D-氨基葡萄糖苷酶(NAGase)活力的影响.结果表明,酶的剩余活力随着DMSO浓度增高而迅速下降,当DMSO浓度达4.0 mol/dm3时,酶活力几乎完全丧失.该酶在低于2 mol/dm3的DM-SO溶液中的失活作用表现为可逆过程.导致酶活力丧失50%的DMSO浓度(IC50)为0.75 mol/dm3.应用酶失活过程的底物反应动力学方法测定了游离酶(E)和酶-底物络合物(ES)在不同浓度DMSO溶液中的微观失活速度常数k+0和k+′0,k+0明显大于k+′0,说明游离酶较酶-底物络合物对DMSO更为敏感,底物对酶被DMSO失活有保护作用.随着DMSO浓度增加,逆向微观复活速度常数k-0不断下降,这说明NAGase在高浓度DMSO中失活的可逆性减弱.

The effect of dimethylsulfoxide （ DMSO ） on N-acetyl-β-D-glucosaminidase （ NAGase ） from green crab （Scylla serrata） has been investigated. The results show that DMSO can inactivate the enzyme activity, and the inactivator＇s concentration leading to 50% of enzyme activity lost （ IC50） is estimated to be 0.75 mol/dm^3. The inactivation of enzyme by DMSO is a reversible reac- tion with remaining enzyme activity. Inactivation kinetics of the enzyme in DMSO solutions is studied using the Tsou＇s method of the substrate reaction, and the microscopic rate constants for the free enzyme and the enzyme-substrate complex are determined and compared. The results show that k＋0 is much larger than k＇ ＋0, indicating a marked protective effect of the substrate on the inactivation reaction of this enzyme by DMSO. Moreover, the value of forward inactivation rate constants of the enzyme （k ＋0 and k＇ ＋0） increased with increasing DMSO concentration while the value of reverse reaction （k-0） decreased. The results suggest that the inactivation of enzyme in higher concentration of DMSO would be much difficultly reversible.