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 hyd rolysis of 10 dansyl peptides by thermolysin. The origin of the fluore scence 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 ther molysin and the dansyl group of the substrate in enzyme-substrate (ES) complexes or from direct excitation (maximal at lambda(ex) 245 and 34 0 nm) of the free substrate and product, or from both sources. These t wo types bf fluorescence signals reflect the concentrations of ES(i) a nd free substrate, respectively. Both types of fluorescence changes ha ve been used to monitor the reaction progress, and different mathemati cal 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 K-M(-1 ) values for the substrates. The quenching efficiency for a dansyl tet rapeptide is weaker due to the greater distance between the dansyl gro up and the Trp-115 donor in thermolysin. On the basis of these studies , substrates capable of supporting more detailed kinetic studies of th ermolysin have been identified.