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 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.
Yang
J J
JJ
Institute of Biochemistry and Cell Biology, Syntex Discovery Research, Palo Alto, California 94304.
Van Wart
H E
HE
D000595:Amino Acid Sequence; D003619:Dansyl Compounds / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013820:Thermolysin / Q000037:antagonists & inhibitors / Q000737:chemistry*
D003619:Dansyl Compounds; D010455:Peptides; D013820:Thermolysin