Tryptophan luminescence from liver alcohol dehydrogenase in its complexes with coenzyme. A comparative study of protein conformation in solution.
The extent of fluorescence quenching and that of phosphorescence quenching of Trp-15 and Trp-314 in alcohol dehydrogenase from horse liver as well as the intrinsic phosphorescence lifetime of Trp-314 in fluid solution have been utilized as structural probes of the macromolecule in binary and ternary complexes formed with coenzyme, analogous, and various substrate/inhibitors. Luminescence quenching by the coenzyme reveals that (1) while the reduced form quenches Trp emission exclusively from the fluorescent state, the oxidized form is very effective on the phosphorescent state as well and that (2) among the series of NADH binary and ternary complexes known by crystallographic studies to attain the closed form, distinct nicotinamide/indole geometrical arrangements are inferred from a variable degree of fluorescence quenching. Information of the dynamic structure of the coenzyme-binding domain derived from the phosphorescence lifetime of Trp-314 points out that within the series of closed NADH complexes there is considerable conformational heterogeneity. In solution, the variability in dynamical structure among the various protein complexes emphasizes that the closed/open forms identified by crystallographic studies are not two well-defined macrostates of the enzyme.
Strambini
G B
GB
CNR, Istituto di Biofisica, Pisa, Italy.
Gonnelli
M
M
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000577:Amides / Q000378:metabolism; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003067:Coenzymes / Q000378:metabolism; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008163:Luminescent Measurements; D015394:Molecular Structure; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011720:Pyrazoles / Q000378:metabolism; D012996:Solutions; D014364:Tryptophan / Q000378:metabolism*
D000577:Amides; D003067:Coenzymes; D011720:Pyrazoles; D012996:Solutions; D009243:NAD; C030197:isobutyramide; D014364:Tryptophan; D000426:Alcohol Dehydrogenase