二甲亚砜对中国鲎NAGase的抑制作用

Inhibitory Effect of Dimethylsulfoxide on N-Acetyl-β-D-Glucosaminidase from Chinese Horseshoe Crab Tachypleus tridentatus

节肢动物的周期性蜕皮与N-乙酰-β-D-氨基葡萄糖苷酶(EC3.2.1.52,NAGase)密切相关。从中国鲎内脏分离提取N-乙酰-β-D-氨基葡萄糖苷酶,利用酶抑制作用动力学方法研究二甲亚砜对中国鲎N-乙酰-β-D-氨基葡萄糖苷酶的影响。试验结果显示:二甲亚砜对中国鲎N-乙酰-β-D-氨基葡萄糖苷酶有较强的抑制作用,0.1 mol/L二甲亚砜可使酶活性下降38.0%,该抑制作用为可逆过程;动力学研究显示,二甲亚砜对N-乙酰-β-D-氨基葡萄糖苷酶的抑制作用是属于竞争性-非竞争性混合型抑制,二甲亚砜对游离酶(E)的抑制常数(K_(I))和对酶-底物络合物(ES)的抑制常数(K_(I)S)分别为0.200 mol/L和1.063 mol/L,K_(I)<K_(I)S,表明底物对N-乙酰-β-D-氨基葡萄糖苷酶在二甲亚砜溶剂中的失活具有保护作用,随着底物浓度增大,二甲亚砜对酶的失活作用降低;酶蛋白的紫外吸收光谱和内源荧光发射光谱显示,随着二甲亚砜溶剂浓度升高,N-乙酰-β-D-氨基葡萄糖苷酶酶蛋白紫外吸收值亦在升高,但其内源荧光发射强度减弱,两者峰值均无位移,在0.65 mol/L二甲亚砜溶剂中酶蛋白荧光强度接近于0,表明N-乙酰-β-D-氨基葡萄糖苷酶在二甲亚砜溶剂中蛋白空间构象发生了变化导致了酶失活现象。试验结果表明,二甲亚砜对中国鲎N-乙酰-β-D-氨基葡萄糖苷酶发挥明显的抑制作用,这可能会进一步影响中国鲎的蜕皮。

The periodic molting of arthropods is closely related to N-acetyl-β-D-glucosaminidase(EC3.2.1.52,NAGase).NAGase was extracted from the viscera of Tachypleus tridentatus.The effect of dimethyl sulfoxide(DMSO)on NAGase was studied by using the kinetics method of enzymatic reaction.The results showed that DMSO had strong inhibitory effect on the NAGase,the enzyme activity decreased by 38.0%when its concentration reached 0.1 mol/L.The inhibitory effect of DMSO on NAGase was reversible.The kinetics showed that the inhibitory effect of DMSO solution on NAGase was the competitive and non-competitive mixed type.The inhibition constants of DMSO on free enzyme(K_(I))and on enzyme-substrate complex(K_(I)S)were determined to be 0.200 mol/L and 1.063 mol/L,respectively.The value of K_(I) was less than that of K_(I)S,indicating a marked protective effect of the substrate on the inactivation of the enzyme.As the concentration of substrate increased,the inactivation of enzyme in DMSO turned to decrease.Meanwhile,UV absorption spectra and fluorescence emission spectra of NAGase were measured.The UV absorption of NAGase treated by DMSO was enhanced with increasing DMSO concentration,but the fluorescence emission peak intensity of the enzyme gradually decreased,their UV absorption peak and fluorescence emission peak of NAGase did not shift.The fluorescence emission of enzyme was almost quenched when the concentration of DMSO reached 0.65 mol/L.It showed that changes of enzyme conformation in the DMSO solvent led to inactivation of the NAGase.The above results confirmed that DMSO might further affect its molting by inhibiting the activity of NAGase from Tachypleus tridentatus.