Fluorescence quenching as an indicator for structural fluctuations in liver alcohol dehydrogenase.
N-Acetyltryptophanamide (NATA), when illuminated anywhere within the 280-nm absorption band, has an emission lifetime of 3.1 ns. The tryptophan residues in liver alcohol dehydrogenase (LADH), however, when excited at 280 nm exhibit two lifetimes of r1 = 2.2 and of r2 = 5.7 ns. Excitation at 300 nm yields a single decay of 5.0 ns. It is shown that at the latter wavelength, only the two (equivalent) tryptophan residues buried within the LADH structure are excited. The reaction rate of the NATA fluorescence quenching by ionic and nonionic quenchers is practically independent of the temperature (between 5 and 41 degrees C). The same substances were used to quench the tryptophan fluorescence in LADH. Here (in the same temperature range), the quenching rate decreases drastically with a decrease in temperature. These findings are discussed in terms of conformational fluctuations in LADH, whereby the temporal movement of the polypeptide chains opens channels through which the above quencher molecules can diffuse and reach the tryptophan residues located within the enzyme structure.
D000429:Alcohol Oxidoreductases; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D011487:Protein Conformation; D011789:Quantum Theory; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000031:analogs & derivatives
D014364:Tryptophan; D000429:Alcohol Oxidoreductases