The Kinetics of Intrinsic Luminescence of Bacterial Luciferase During Catalysis Indicates Flavin Binding and Enzyme Recovery

The study proposes an experimental approach to determining the rates of individual stages of a reaction catalyzed by bacterial luciferase, based on the tryptophan fluorescence of the protein and the stopped-flow technique. The relationship between the fluorescence intensity of tryptophan residues in luciferase and the presence of substrates and reaction products in the active site of the enzyme is substantiated. The non-steady-state kinetics of bioluminescence in the reaction of two bacterial luciferases with aliphatic aldehydes of different chain lengths, as well as the kinetics of enzyme fluorescence intensity during the reaction, were analyzed. The obtained results confirmed the relationship between the rate of the two kinetic stages of enzyme luminescence and the processes of flavin substrate binding and enzyme activity recovery after the catalytic act.