Kinetics of Hydrolysis of Dansyl Peptide Substrates by Thermolysin: Analysis of Fluorescence Changes and Determination of Steady-State Kinetic Parameters

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...

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Bibliographic Details
Published in:Biochemistry (Easton) 1994-05, Vol.33 (21), p.6508-6515
Main Authors: Yang, Jenny Jie, Van Wart, Harold E
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Dansyl Compounds - chemistry
Hydrolysis
Kinetics
Molecular Sequence Data
Peptides - chemistry
Peptides - metabolism
Spectrometry, Fluorescence
Substrate Specificity
Thermolysin - antagonists & inhibitors
Thermolysin - chemistry
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00187a018