甲基对硫磷水解酶参与催化相关结构的研究  

Determination of the Catalytic Structures of Methyl Parathion Hydrolase

作　　者：吴旭平[1] 刘卫东[1] 曹慧[1] 李顺鹏[1] 崔中利[1] 

机构地区：[1]南京农业大学农业部农业环境微生物工程重点开放实验室,南京210095

出　　处：《生物工程学报》2005年第6期998-1002,共5页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30300005和40371069);国家高技术研究与发展(No.2004AA246070)项目资助。~~

摘　　要：甲基对硫磷水解酶(MPH)是一种新的有机磷水解酶。将完整的甲基对硫磷水解酶基因(mpd)构建入pUC19载体,使得mpd基因以自身的启动子在EscherichiacoliDH5α中表达并得到了纯化。金属螯合实验发现MPH的活性不受金属螯合剂1,10菲啉的影响;但用电感耦合等离子发射光谱测定其金属含量显示MPH是金属酶,1mol酶中结合了2mol的Zn2+。为确定参与MPH催化活性的必需氨基酸,用化学修饰剂碳化二亚胺、二乙基焦磷酸酯、磷酸吡哆醛和丁二酮处理MPH,然后检测其残余酶活力,结果表明天冬氨酸、谷氨酸、赖氨酸和精氨酸残基与酶的催化活性无关;而二乙基焦磷酸酯对组氨酸侧链的化学修饰引起酶活性的大幅度的下降,其对酶活性的抑制率达到9.6h-1,说明组氨酸是酶活力所必需的基团。这些结果为进一步研究酶的结构及对酶进行分子改造提供了必要的基础数据。Methyl parathion hydrolase （MPH） is a novel member of organophosphorus hydrolase. In this study, mpd gene was expressed in Escherichia coli DH5α with its native promoter. MPH was purified to homogeneity. Results show that metalchelating compounds cannot inhabit the enzyme activity. Inductively Coupled Plasma-Atomic Emission Spectrometry analysis showed that MPH is a zinc-containing enzyme, the Zinc to enzyme molar ratio is near 2 ： 1. In order to investigate critical residues related to enzymatic activity of MPH, chemical modification reagents EDC, DEPC, butanedione and pyridoxal were tested. Experiment results suggested that aspartate, glutamate, arginine and lysine are not important for enzyme activity. But DEPC, which can modify histidine residue, inactivate the enzyme activity greatly, and the inactivation rate is 9.6h^-1. This result reflects that histidine residues are essential for enzyme activity. All these results provide basic data for MPH structure and molecular evolution research.

关 键 词：甲基对硫磷水解酶 金属离子含量 化学修饰 必需氨基酸 

分 类 号：Q936[生物学—微生物学]