【摘要】 为了解蛋白质变性剂对酵母蔗糖酶（YInv）及其修饰酶（COS-Inv）的作用效果及分子机理,用尿素和盐酸胍处理天然蔗糖酶及其修饰酶,对处理前后酶活性的变化、酶的催化动力学、吸收光谱、紫外差示光谱和荧光光谱进行了探索。研究结果发现,低浓度(<0.3 mol·L^-1)尿素对COS-Inv有激活作用,0.2 mol·L^-1的尿素溶液使其活性提高了10.54%;但尿素对YInv只表现出抑制作用。盐酸胍对YInv和COS-Inv的活性都产生明显的抑制作用。动力学分析结果显示,0.2 mol·L^-1的尿素可提高COS-Inv与底物的亲和力,而降低YInv与底物的亲和力。盐酸胍对YInv的抑制主要是通过降低最大反应速度来实现的,其反应机理类似于反竞争性抑制。盐酸胍对COS-Inv的抑制是通过降低酶与底物的亲和力来实现的,类似于竞争性抑制。紫外吸收光谱、紫外差示光谱和荧光光谱分析显示,在盐酸胍和尿素溶液中,YInv和COS-Inv的分子构型基本不变,但其构象却发生明显变化。更多还原

【Abstract】 To comprehend the effects of protein denaturants on the yeast invertase（YInv）and its modified enzyme（COS-Inv） and the involved molecular-level action mechanism, we used urea and guanidine hydrochloride to treat YInv and COS-Inv. The changes of enzyme activities,catalytic kinetics,absorption spectra,ultraviolet differential spectra and fluorescence spectra of YInv and COS-Inv were investigated. The results showed that lower concentration(<0.3 mol·L^-1)of urea activates COS-Inv. 0.2 mol·L^-1 urea solution increases the activity of COS-Inv by 10.54%. However,urea only shows inhibitory effect on YInv. The activities of both YInv and Cos-Inv are significantly inhibited by guanidine hydrochloride. Kinetic analysis results showed that 0.2 mol·L^-1 urea increases the affinity between COS-Inv and the substrate,while decreases the affinity between YInv and the substrate. The inhibition of YInv by guanidine hydrochloride is mainly achieved by reducing the maximum reaction velocity. The reaction mechanism was similar to anti-competitive inhibition. The inhibition of COS-Inv by guanidine hydrochloride is achieved by reducing the affinity between the enzyme and the substrate,which is similar to competitive inhibition. The analysis of UV absorption spectra,UV differential spectra and fluorescence spectra showed that the molecular configuration of YInv and COS-Inv is basically unchanged in the solutions of guanidine hydrochloride and urea,but their conformation has changed dramatically.更多还原