Quenching of the Fluorescence of Tyr and Trp Residues of Firefly Luciferase from Luciola mingrelica by the Substrates

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T. N. Vlasova^* and N. N. Ugarova

Faculty of Chemistry, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-2660; E-mail: vtn7@yandex.ru

^* To whom correspondence should be addressed.

Received April 17, 2007; Revision received May 23, 2007

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Luciferase of the firefly Luciola mingrelica is characterized by fluorescence of not only the unique Trp residue (lambda_em = 340 nm), but also that of Tyr residues (lambda_em = 308 nm). Quenching of the intrinsic fluorescence of the luciferase by its substrates luciferin and ATP (AMP) has been studied. Luciferin (LH₂) quenches Trp fluorescence more efficiently than the fluorescence of Tyr residues. Two centers of quenching of Tyr fluorescence by ATP have been found corresponding apparently to the allosteric and active sites of the luciferase with K_s(ATP) = 20 and 110 µM, respectively. The influence of one substrate on the affinity of luciferase to the second was investigated using fluorescence. ATP (AMP) binding to the allosteric sites of the luciferase significantly affects the affinity of luciferase to LH₂. Formation of the complex between the luciferase and LH₂ affects the affinity of both allosteric and active sites of the luciferase to ATP (AMP). The observed effects are probably connected with conformational changes in the luciferase molecule upon its interaction with the substrates.

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KEY WORDS: firefly luciferase, bioluminescence, Tyr fluorescence, Trp fluorescence, luciferin, ATP, AMP

DOI: 10.1134/S0006297907090064

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