Hg2+和Pb2+对中国鲎N-乙酰-β-D-氨基葡萄糖苷酶的影响

Effects of Hg2+ and Pb2+ on N-acetyl-β-D-glucosaminidase of Tachypleus tridentatus

节肢动物蜕皮与N-乙酰-β-D-氨基葡萄糖苷酶(EC3.2.1.52,NAGase)密切相关。为了探究Hg^2+和Pb^2+这2种重金属离子对节肢动物NAGase的影响,以中国鲎(Tachypleus tridentatus)为材料,从其内脏分离提取了NAGase。利用酶促反应动力学方法,研究Hg^2+和Pb^2+对中国鲎NAGase的影响;通过对酶荧光发射光谱的测定,研究NAGase酶蛋白经Hg^2+和Pb^2+作用后的空间构象变化。结果表明,Hg^2+和Pb^2+对中国鲎NAGase均有较强的抑制作用,Hg^2+对NAGase的抑制作用强于Pb^2+,Hg^2+和Pb^2+对NAGase的抑制作用均表现为可逆过程,其中Hg^2+对NAGase的抑制属于竞争性抑制作用,抑制常数K I为22.68μmol·L^-1,Hg^2+只与游离酶结合,不与酶-底物络合物结合;而Pb^2+对NAGase的抑制是属于竞争性-非竞争性混合型抑制作用,抑制常数K I和K IS分别为19.13 mmol·L^-1和75.23 mmol·L^-1,Pb^2+与游离酶的亲和力相比与酶-底物络合物的亲和力更强。NAGase经Hg^2+和Pb^2+作用后荧光发射强度均降低,但荧光发射峰值并没有发生位移,说明Hg^2+和Pb^2+对中国鲎NAGase的抑制作用均为酶蛋白空间构象的变化所致。这证明了Hg^2+和Pb^2+对中国鲎NAGase活力具调控作用。

The molting of arthropods is closely related to N-acetyl-β-D-glucosaminidase(EC3.2.1.52,NAGase).In order to investigate the effects of Hg^2+and Pb^2+heavy metal ions on NAGase from arthropods,the NAGase were extracted from the viscera of Tachypleus tridentatus.The effects of Hg^2+and Pb^2+on NAGase were studied by using the kinetics method of enzymatic reaction.Besides,the conformational changes of NAGase caused by Hg^2+and Pb^2+treatment were evaluated by the enzyme fluorescence emission spectrometry,respectively.The results showed that both Hg^2+and Pb^2+had strong inhibitory effects on the NAGase.The inhibitory effect of Hg^2+on NAGase was stronger than that of Pb^2+.The inhibitory effects of Hg^2+and Pb^2+on NAGase were both reversible.The inhibitory effect of Hg^2+on NAGase was the competitive type and its inhibition constant K I was 22.68μmol·L^-1.Hg^2+only combined with the free enzymes(E)and did not combine with the enzyme-substrate complex(ES).However,the inhibitory effect of Pb^2+on NAGase was the competitive and non-competitive mixed type.The inhibition constants K I and K IS were 19.13 mmol·L^-1 and 75.23 mmol·L^-1,respectively.The affinity of Pb^2+for free enzyme was stronger than the enzyme-substrate complex.The fluorescence emission intensity of the NAGase treated by Hg^2+and Pb^2+were both decreased,but their fluorescence emission peak of NAGase did not shift.The results indicated that the inhibitory effects of both Hg^2+and Pb^2+on NAGase were caused by the conformational changes,which suggested that Hg^2+and Pb^2+had regulatory effects on the activities of NAGase from Tachypleus tridentatus.