【摘要】 通过量子化学计算,确定嗜热菌Pyrococcus horikoshii OT3的PH1704蛋白酶别构位点的关键残基为Arg113,Tyr120和Asn129.其中,Arg113及Asn129与别构抑制剂结合,参与别构调控.Tyr120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k cat/k m(L·μmol-1·min-1)值是野生型k cat/k m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α7螺旋(124~129,524~529)间聚合度增强;120位点残基由Tyr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.更多还原

【Abstract】 The PH1704 allosteric sites were studied with the quantum chemistry analysis and the crystal structure analysis.The results show that key residues are Arg113,Tyr120 and Asn129.Tyr120 is connected with nucleophilic residues Cys100 by a hydrogen bond,participates in enzyme nucleophilic catalyst,and is validated 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 k cat/k m(L·μmol- 1·min- 1) value of mutant R113T/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 Tyr120 and Cys100 are broken,thus its nucleophilic attacking resistance decreases,which causes the enzyme activity to increase.The mutations of 113 and 129 sites lead to the detachment of the anionic allosteric agent,thus the negative cooperativity disappears.This work predictes the allosteric 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.更多还原