醋酸酐对太平洋白对虾(Penaeus vannamei)β-N-乙酰-D-氨基葡萄糖苷酶的抑制动力学(英文)

Inhibition Kinetics of Acetic Anhydride on Enzyme Activity of β-N-Acetyl-D-glucosaminidase from Pacific White Shrimp(Penaeus vannamei)

β-N-乙酰-D-氨基葡萄糖苷酶与太平洋白对虾的食物消化吸收、蜕壳生长有着密切关系.海水里存在的有机污染物将影响酶生理功能,从而进一步影响虾的正常蜕壳,严重将导致对虾的死亡.醋酸酐是常用的有机溶剂,故本文应用动力学方法研究醋酸酐对太平洋白对虾β-N-乙酰-D-氨基葡萄糖苷酶催化pNP-NAG水解时酶活力的变化规律.表明在醋酸酐浓度低于20.0mmol/L,酶的抑制作用是可逆的,测得醋酸酐对酶抑制的IC50为9.0mmol/L.用双倒数作图法测定醋酸酐与游离酶(E)和酶-底物络合物(ES)的结合平衡常数.结果显示,醋酸酐是酶的非竞争性抑制剂.用底物反应动力学方法观测在不同底物浓度下酶在0.0、3.0、6.0、9.0、12.0mmol/L的醋酸酐溶液中的失活过程,分别测定了酶的微观失活速度常数k+0及复活速度常数k-0,结果表明醋酸酐对酶的影响是快速结合再缓慢失活的过程.比较微观失活速度常数k+0及复活速度常数k-0,结果显示,在高浓度的醋酸酐溶液中,酶将完全失活.

β- N-Acetyl- D-glucosaminidase （ NAGase, EC3.2.1.52 ） is a composition of the chitinases and cooperates with endo-chitinase and exo-chitinase to disintegrate chitin into N-acetylglucosamine. Pacific White Shrimp （P. vannamei ） NAGase is involved in digestion and molting processes. Some pollutants in seawater affect the enzyme activity causing loss of the biological function of the enzyme, which affects the exuviating shell and threatens the survival of the animal. The effects of acetic anhydride on the enzyme activity for the hydrolysis of pNP-NAG have been studied. The results show that acetic anhydride can lead to reversible noncompetitive inhibition at appropriate concentrations, and the IC50 is estimated to be 9.0 mmol/L. The equilibrium constants have been determined for acetic anhydride binding with the enzyme and/or the enzymesubstrate complexes. Inhibition kinetics of acetic anhydride on the enzyme has been studied using the kinetic method of the substrate reaction. The results suggest that at pH 6.2, the action of acetic anhydride on the enzyme is first quick equilibrium binding and then slow inhibition. The microscopic rate constants have been determined for inhibition and reactivation. The results show that k ＋ 0 is much larger than k -0, indicating the enzyme is completely inactivated at sufficiently large modificator concentration.