AbstractThe stopped‐flow fluorescence traces for the hydrolysis of Leu‐Ala‐DED catalyzed byStreptomyces griseusaminopeptidase isolated from pronase exhibits a two‐phase fluorescence change with comparable rates of formation and breakdown of the ES complex. We have developed a method for the determination of the individual rate constants from the stopped‐flow traces. The kinetic parameters for the enzyme‐catalyzed hydrolysis of Leu‐Ala‐DED at 25 °C and pH 8.0 are:k1= (1.6±0.1) × 105M−1s−1,k−1, = 0.089 ± 0.001 s−1, andk2= 0.58 ± 0.01 s−1. For the observed stopped‐flow traces, the steady state approximation is valid only within a very small region around the maximal ES concentration and a large proportion (>20%) of the substrate has already been hydrolyzed during the pre‐steady state. For a very fast formation of ES complex, the steady state approximation is valid for almost the entire trace. The activation energies for each individual rate constant were determined to be 10.0 ± 0.7, 35 ± 5, and 21.2 ± 0.8 kcal mol−1fork1, k−1, and k2, respectively. Binding of E and S to form ES was accompanied by a decrease in Gibbs free energy, whereas a dramatic increase in free energy was observed for the conversion of ES to ES≠. The dissociation of ES to form E and S had a very large activation energy, but it was also