Quenching of tryptophan fluorescence of firefly luciferase by substrates

The interaction of firefly luciferase with substrates (luciferin and MgATP) by steady-state and time-resolved fluorescence is studied. The efficient q uenching of tryptophan fluorescence of the active enzyme takes place upon i ts binding with substrates. In the presence of ATP the quenching is of dyna mic type and is caused by structural changes in the protein molec;le upon A TP binding. A model is proposed in which the complex has smaller fluorescen ce quantum yield than the free enzyme because of partial quenching of trypt ophan fluorescence by the new microenvironment. Quenching of tryptophan flu orescence by luciferin due to the efficient energy transfer from tryptophan to luciferin is discussed. The calculated distance between Trp-419 and luc iferin for the L. mingrelica luciferase in the enzyme-substrate complex is less than 12 Angstrom. (C) 2001 Elsevier Science B.V. All rights reserved.