纤维素酶活性架构是酶分子中多个氨基酸残基构成的可结合并催化底物的功能区,其中色氨酸等芳香族残基在该区域中起着重要作用.本研究利用荧光光谱法,定量分析了纤维素酶Ch Cel5A活性架构中色氨酸与底物的结合动力学过程,通过色氨酸荧光猝灭的定量分析,确定了色氨酸特异性结合时的底物浓度范围,并且测定了Ch Cel5A活性架构中单个氨基酸突变导致的底物结合常数的变化,与催化动力学参数比较发现,荧光光谱法可准确表征纤维素酶与底物的结合力及其单个残基突变引起动力学参数的变化.此外,由于p NP中含有强的吸电子基团,因而以p NPC等为配体时会高估与色氨酸的结合常数约20-100倍.荧光光谱法可以测定纤维素酶结合糖分子底物的动力学参数,该方法具有灵敏和快速的特点,这为蛋白质与底物之间相互作用的定量分析提供了新的视角.
The active-site architecture of cellulase is a functional area composed of multiple amino acid residues which can bind and catalyze substrates. Aromatic residues, such as tryptophan, play important roles in the function of this region. In this study, the dynamic of binding process between tryptophan in active-site architecture of cellulase Ch Cel5 A and substrate was quantitatively analyzed using fluorescence spectrum. The substrate concentration range when tryptophan can specifically bind the substrate was determined and the change of binding constant caused by a single amino acid mutation in active-site architecture of Ch Cel5 A was measured through quantitative analysis of tryptophan fluorescence quenching. Compared with the measurement of kinetic parameters,fluorescence spectroscopy could accurately characterize the binding affinity between cellulase and substrate, as well as the change of kinetic parameter caused by a single residue mutation. Besides, when using p NPC as the ligand, the binding constant could be overestimated for 20-100 times due to the increase of strong electrophilic groups. Fluorescent spectrometry can measure the kinetic parameter of cellulase-substrate binding process in a sensitive and rapid way, providing a new perspective for the quantitatively analyzing the interaction between proteins and substrates.