色氨酸残基在碳-碳水解酶催化中的关键作用  

Tryptophan 266 is Required for the C-C Hydrolase Activity

作　　者：杨秀清[1] 沈翀[1] 高冲[1] 

机构地区：[1]山西大学生物技术研究所,化学生物学与分子工程教育部重点实验室,太原030006

出　　处：《中国生物化学与分子生物学报》2012年第6期538-545,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.30800030);山西省青年科技基金(No.207021030)资助~~

摘　　要：碳-碳水解酶(C-C水解酶)作为α/β水解酶超家族中的一员,负责催化环裂产物C-C键的断裂,该反应是细菌降解芳香族化合物途径中的关键步骤.为了解水解酶的催化特性,本文对该酶部分氨基酸进行了定点突变,并对突变体的动力学参数,化学修饰剂对突变体活性的影响以及突变体的二级结构进行了测定.各突变体的动力学参数特征为:突变体S110A,H265A和D237A的催化效率为野生型的1/104～1/103;突变体W85A和W219A催化效率分别为野生型的5/18和1/3,而同为色氨酸的突变体,W266A的催化效率只有野生型的1/104.化学修饰剂对突变体S110A,H265A,D237A和W266A的酶活性几乎没有影响;而对突变体W85A和W219A却有较大的影响,修饰后,其相对活性仅为对照的10%～30%.突变体的圆二色谱(CD谱)分析表明,与野生型相比,突变体的二级结构没有发生改变.证明了Ser110,Asp237,His265是2-羟基-6-氧-6-苯基己-2,4-二烯酸水解酶(HOPDA hydrolase,HOPDA水解酶)催化反应所必需的氨基酸,并提出了Trp266在催化反应中也同样起到了非常关键的作用.As a member of the α/β-hydrolase superfamily, Rhodococcus sp. R04 derived BphD C-C hydrolase （2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate, HOPDA hydrolase） catalyses the hydrolytic C-C cleavage of the meta-ring fission products from the biphenyl catabolic pathway. We constructed six mutants of C-C hydrolase to investigate its catalytic mechanism. Substitutions of Ser-ll0, Asp-237 and His-265 in HOPDA hydrolase with Ala caused to 17 - 19-fold reduction in kcat and reduced the kcat/Km values by 10^4 10^3folds in comparison to the wild-type. The Ala substitution of Trp-85 and Trp-219 led to 3.6- and 3.3-fold reduction in kcat/Km, respectively. Mutating of Trp-266 to Ala resulted in a 104-fold reduction of kcat/Km. From the circular dichroism （CD）spectra, Sll0A, D237A, H265A and W266A were predominately showed in a-helix conformation as the wild-type. The chemical modification results showed that the modifiers did not influence the activities of Sll0A, D237A, H265A and Trp266 mutants, except 0.5 mmol/L NBS reduced the activity of W85A by 90% and W219A by70%. Our data indicated that Ser110, Asp237 and His265 were necessary for the catalytic reaction of HOPDA hydrolase, and Trp266 was suggested to play a key role in the enzymatic reaction.

关 键 词：色氨酸 碳-碳水解酶 定点突变 化学修饰 圆二色谱(CD谱) 

分 类 号：Q936[生物学—微生物学]