Yang J J, Van Wart H E
Institute of Biochemistry and Cell Biology, Syntex Discovery Research, Palo Alto, California 94304.
Biochemistry. 1994 May 31;33(21):6508-15. doi: 10.1021/bi00187a018.
The stopped-flow fluorescence technique has been used to study the hydrolysis of 10 dansyl peptides by thermolysin. The origin of the fluorescence changes observed during the reactions has been investigated in detail. Depending on the substrate and the excitation wavelength, the dansyl fluorescence changes observed arise either from energy transfer (maximal at lambda ex = 230 and 280 nm) between Trp residues of thermolysin and the dansyl group of the substrate in enzyme-substrate (ES) complexes or from both sources. These excitation (maximal at lambda ex = 245 and 340 nm) of the free substrate and product, or from both sources. These two types of fluorescence signals reflect the concentrations of ESi and free substrate, respectively. Both types of fluorescence changes have been used to monitor the reaction progress, and different mathematical formalisms have been used to determine the kinetic parameters for the reactions with results that are in good agreement. The efficiency of Trp quenching by a series of five dansyl tripeptides is shown to be related to the fractional saturation of enzyme and follows the KM-1 values for the substrates. The quenching efficiency for a dansyl tetrapeptide is weaker due to the greater distance between the dansyl group and the Trp-115 donor in thermolysin. On the basis of these studies, substrates capable of supporting more detailed kinetic studies of thermolysin have been identified.
停流荧光技术已被用于研究嗜热菌蛋白酶对10种丹磺酰肽的水解作用。已详细研究了反应过程中观察到的荧光变化的来源。根据底物和激发波长的不同，观察到的丹磺酰荧光变化要么源于嗜热菌蛋白酶的色氨酸残基与酶 - 底物（ES）复合物中底物的丹磺酰基团之间的能量转移（在λex = 230和280 nm处最大），要么源于这两种来源。这些变化来自游离底物和产物的激发（在λex = 245和340 nm处最大），或者来自这两种来源。这两种类型的荧光信号分别反映了ESi和游离底物的浓度。这两种类型的荧光变化都已用于监测反应进程，并且使用了不同的数学形式来确定反应的动力学参数，结果吻合良好。一系列五种丹磺酰三肽对色氨酸的淬灭效率显示与酶的分数饱和度相关，并遵循底物的KM-1值。由于丹磺酰基团与嗜热菌蛋白酶中色氨酸 - 115供体之间的距离更大，丹磺酰四肽的淬灭效率较弱。基于这些研究结果，已鉴定出能够支持对嗜热菌蛋白酶进行更详细动力学研究的底物。
