嗜热蛋白酶PH1704别构中心量子化学计算与突变体动力学  

Quantum Chemistry Calculation of Thermophilic Protease PH1704 Allosteric Center and Mutant Dynamics

作　　者：詹冬玲[1,2] 高楠[3] 韩葳葳[1] 冯雁[1] 

机构地区：[1]吉林大学分子酶学工程教育部重点实验室,长春130012 [2]吉林农业大学食品科学与工程学院,长春130118 [3]中国科学院长春应用化学研究所,长春130022

出　　处：《高等学校化学学报》2014年第1期146-153,共8页Chemical Journal of Chinese Universities

基　　金：国家'九七三'计划项目(批准号:2012CB721003);国家自然科学基金(批准号:31070638);吉林省自然科学基金(批准号:201015109)资助~~

摘　　要：通过量子化学计算，确定嗜热菌PyrococcushorikoshiiOT3的PHl704蛋白酶别构位点的关键残基为Argll3，Tyrl20和Asnl29．其中，Argll3及Asnl29与别构抑制剂结合，参与别构调控．Tyrl20残基位于亚基交界面附近，并与亲核残基Cys100之间以氢键相连，可通过影响亚基聚合来影响酶的亲核催化．DJ-1超家族的4种构建蛋白的结构显示，120位点位于亚基交界面处，影响亚基的聚合，进而影响蛋白酶的活力，并间接参与别构调控．分子生物学实验显示，突变体R113T／Y120P／N129D的kcat/km（L·μmol^-1·min^-1）值是野生型kcat/km。值的6倍，h系数由野生型的0．86转变为1．3，负协同效应消失．113和129位点处阴离子别构剂脱离，从而破坏113，120和129位点间的封闭环结构，使AC交界面胡螺旋（124～129，524—529）间聚合度增强；120位点残基由rryr转变为Pro，与Cys100间氢键断裂，亲核进攻的阻力减小，从而使酶活力提高，别构负调控消失．The PH1704 allosteric sites were studied with the quantum chemistry analysis and the crystal struc- ture analysis. The results show that key residues are Arg113, Tyr120 and Asn129. Tyrl20 is connected with nucleophilic residues Cysl00 by a hydrogen bond, participates in enzyme nucleophilic catalyst, and is valida- ted by fixed-point mutation of molecular biology experiments. The structures of four building protein of DJ-1 superfamily show that the 120 site locates in the substrate binding pocket in the subunit interface and affects the enzyme activity of the protein. The kcat/km（ L · μmol-1 · min-1） value of mutant Rl13T/Y120P/N129D is six times higher than that of the wild-type and the Hill coefficient changes from 0. 86 （ wild type） to 1.3 with negative cooperativity disappearing. The main reason is that the residue of 120 site changes from Tyr to Pro, and the hydrogen bonds between Tyrl20 and Cysl00 are broken, thus its nucleophilic attacking resis-tance de- creases, which causes the enzyme activity to increase. The mutations of 113 and 129 sites lead to the detach- ment of the anionic allosteric agent, thus the negative eooperativity disappears. This work predictes the allos- teric site of thermophilic protease by quantum chemistry and crystal structure analysis and provides a solid foundation for further research on the allosteric enzyme of DJ-1 superfamily.

关 键 词：嗜热蛋白酶 量子化学计算 别构中心 定点突变 

分 类 号：O641[理学—物理化学] O623[理学—化学]