枯草杆菌及大肠杆菌异柠檬酸脱氢酶的酶学性质研究  

Enzymatic Characterization of Isocitrate Dehydrogenases from Bacillus subtilis and Escherichia coli

作　　者：许希晨[1] 葛亚东[1,2] 王敖[1] 王程[1] 朱国萍[1,2] 

机构地区：[1]安徽师范大学生命科学学院分子进化与生物多样性重点实验室,安徽芜湖241000 [2]安徽师范大学生命科学学院分子生物学及生物技术研究所,安徽芜湖241000

出　　处：《激光生物学报》2010年第3期373-379,396,共8页Acta Laser Biology Sinica

基　　金：国家自然科学基金项目(30500300;30870062);教育部新世纪优秀人才支持计划项目(NCET-06-0558);安徽省优秀青年科技基金项目(06043089);安徽省引进海外留学人才基金项目(2005Z032)

摘　　要：对枯草杆菌异柠檬酸脱氢酶(BsIDH)、大肠杆菌异柠檬酸脱氢酶(EcIDH)和大肠杆菌异柠檬酸脱氢酶的突变体酶(EmIDH)进行了纯化和酶学性质鉴定。BsIDH和EcIDH对辅酶NADP^+的特异性与NAD^+相比,分别是NAD^+的1 330倍和3 890倍。而EmIDH对NAD^+的特异性与NADP^+相比,是NADP^+的122倍。因此,BsIDH和EcIDH是NADP^+依赖性异柠檬酸脱氢酶,而EmIDH的辅酶特异性已转换为NAD^+依赖性。EcIDH、BsIDH和EmIDH对底物异柠檬酸的K_m值分别为67.4 μmol/L、60.6μmol/L和105.6μmol/L。BsIDH和EcIDH的最适反应pH分别为8.2和8.0,EmIDH的最适反应pH为7.0。BsIDH和EmIDH的最适反应温度是45℃,EcIDH的最适温度为43℃。三种IDH的活性依赖于不同的二价金属离子的存在,Mn^(2+)、Mg^(2+)存在时酶活性最强,Cu^(2+)、Ca^(2+)、Zn^(2+)和Ni^(2+)强烈抑制酶的活性。系统的酶学性质研究为深入认识IDH的催化与调节机制提供了更多依据。The Bacillus subtilis isocitrate dehydrogenase （BsIDH）, the Escherichia coli wild-type isocitrate dehydrogenase （EcIDH） and its engineered mutant isocitrate dehydrogenase （EmIDH） were purified and their enzymatic properties were characterized. Coenzyme specificities （calculated by the specific value of kcat/Km for NADP^＋ and NAD^＋） of BsIDH and EcIDH were 1330-fold and 3890-fold greater for NADP^＋ than NAD^＋, respectively. The mutant EmIDH showed 122-fold preference for NAD^＋ over NADP^＋. Both of BsIDH and EcIDH were typical NADP^＋-dependent IDHs, while EmIDH had been changed to be a NAD^＋-dependent enzyme. The Km for isocitrate of BsIDH, EcIDH and EmIDH were 60.6 μM, 67.6 μM and 105.6 μM, respectively. The pH optimum of BsIDH, EcIDH and EmIDH were 8.2, 8.0 and 7.0, and their optimal temperatures were 45 °C, 45 °C and 43 °C, respectively. The activity of three IDHs was entirely dependent on the binding of Mn^2＋ or Mg^2＋. The maximum activity of IDHs was observed when assayed with Mn^2＋ or Mg^2＋ as metal cofactor. Cu^2＋, Ca^2＋, Zn^2＋ and Ni2＋ dramatically inhibited the activity of IDHs. The comprehensive information of enzymatic properties may provide the further insight into the mechanism of catalysis and function of isocitrate dehydrogenase.

关 键 词：枯草杆菌 大肠杆菌 异柠檬酸脱氢酶 突变体酶 辅酶特异性 酶学性质 

分 类 号：Q786[生物学—分子生物学]