Kinetics of hydrolysis of dansyl peptide substrates by thermolysin: analysis of fluorescence changes and determination of steady-state kinetic parameters.
Kinetics of hydrolysis of dansyl peptide substrates by thermolysin: analysis of fluorescence changes and determination of steady-state kinetic parameters.
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嗜热菌蛋白酶水解丹酰肽底物的动力学：荧光变化分析和稳态动力学参数的测定。
DOI：
10.1021/bi00187a018
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
VanWart,HE
中科院分区：
文献类型：
--
作者：
Yang,JJ;VanWart,HE
Revised Manuscript Received March 15, 1994® abstract: The stopped-flow fluorescence technique has been used tostudy 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 ß=230 and 280 nm) between Trp residues of thermolysin and the dansyl group of the substrate in enzyme-substrate (ES) complexes or from direct excitation (maximal at Xex= 245 and 340 nm) of the freesubstrate and product, or from both sources. These two types of fluorescence signals reflect the concentrations of ES,· 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 ATm-1 values for thesubstrates. 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.Thermolysin (EC 3.4. 24.4) is a thermostable zinc proteinase from Bacillus thermoproteolyticus (Endo, 1962). The enzyme has been extensively studied and is believed to share many critical mechanistic features with mammalian metallopepti-dases such as carboxypeptidase A, angiotensin converting enzyme, and the matrix metalloproteinases. The enzyme contains one zinc atom (Latt et al., 1969) located at the active site that participates in catalysis. It also contains four atoms of calcium that confer thermal stability by stabilizing its
