快速展示糖苷水解酶活性架构单点突变导致结合力变化的电泳技术

Affinity Electrophoresis for The Rapid Identification and Characterization of Substrate Binding Ability Influenced by Amino Acid Single-Point Mutation in Active-Site Architecture of Glucoside Hydrolase

酶分子在长期进化过程中形成一系列氨基酸残基组成的活性架构,参与底物的识别、结合与催化过程,而活性架构中相应氨基酸残基是如何影响酶分子结合底物的能力,进而影响酶分子的催化效率,一直是酶分子理性改造研究的热点.利用亲和电泳技术,可以快速展示内切纤维素酶Tr Cel12A和木聚糖酶Tl Xyn A活性架构中不同突变体的催化活性及其迁移率的变化,进而通过在不同底物浓度凝胶中蛋白质相对迁移率变化程度的定量回归分析,发现由氨基酸单点突变导致蛋白质迁移率的相对变化,可以定量表征酶分子突变前后结合底物能力的变化.亲和电泳测定的有效阻滞常数Kb值与等温滴定量热法和荧光光谱法测定的相关参数比较具有明显相关性.由于亲和电泳技术在测定酶分子与底物的结合能力时具有简便、快速、灵敏的特点,因而可作为常规生化实验室常规普筛技术来检测突变文库中系列突变体导致结合力的变化.

The enzyme molecule is natural effective biological catalyst. The enzyme-substrate（ES） complex forming in the first step imparts the catalytic efficiency of enzyme. Specifically, multiple amino acid residues composed the active site of enzyme, which created a precisely organized architecture for substrate recognition,binding and catalytic process. Therefore, the function of amino acid residues in active-site architecture has always been the research hotspot in protein engineering. The enzymes in GH11 and GH12 families have small molecular mass and large range of enzymology properties, which are good candidates to study the binding affinity of enzymes. In this study, the catalytic activity and band mobility of different mutants in the active-site architecture of Tr Cel12 A and Tl Xyn A could be rapidly demonstrated by affinity electrophoresis. The substrate binding affinity can be quantitatively characterized by quantitative regression analysis of the relative mobility of proteins at different substrate concentrations. At the same time, isothermal titration calorimetry and fluorescence spectroscopy were also used to determine the binding affinity of different mutants. For the mutants of Tr Cel12 A, the parameters Kb measured by affinity electrophoresis were significantly related with those determined by isothermal titration calorimetry（R2= 0.82） and fluorescence spectroscopy（R2= 0.83）. Therefore, the magnitude of their affinities was Tr Cel12 A-E200 QW22 YE200 QW22 FE200 QW22 HE200 QW22 AE200 Q, respectively. Furthermore, the substrate binding affinities of the variants in Tl Xyn A were also characterized using affinity electrophoresis. The results showed that there was a good correlation with that measured by fluorescence spectrometry（R2= 0.84）.These results indicate that the parameters Kbmeasured by affinity electrophoresis can characterize the binding ability influenced by amino acid single-point mutation in active-site architecture of glucoside hydrolase. Therefore,affinity electrophoresis was effective and can be used as routine screening technology in biochemical laboratory to detect the change of series mutants binding affinity in mutant library.