Characterization of tryptophan environments in glutamate dehydrogenases from temperature-dependent phosphorescence.
Tryptophan room temperature phosphorescence in solution was detected in glutamic dehydrogenase from bovine liver and Escherichia coli with lifetimes of 1.2 and 0.65 s, respectively. Although these enzymes possess three and five tryptophanyl residues per polypeptide chain, respectively, the temperature dependence of the phosphorescence quantum yield estimates that the room temperature emission is due, in either case, to a single residue. Long triplet-state lifetimes and very small rates of O2 quenching indicate that these tryptophanyl side chains are embedded in a highly inflexible internal region of the macromolecule. Aided by sequence homology with dehydrogenases of known structure and theoretical predictions of secondary structure [Wootton, J.C. (1974) Nature (London) 252, 542-546; Brett, M., Chambers, G.K., Holder, A. A., Fincham, J.R.S., & Wootton, J.C. (1976) J. Mol. Biol. 106, 1-22], the phosphorescing tryptophans have been tentatively placed in the catalytic coenzyme binding domain of each enzyme. The particular sensitivity of the triplet-state lifetime in probing local changes in conformation provides a strong indication that within the time window of phosphorescence measurements the six subunits in the hexameric enzymes are equivalent. Furthermore, while in the bovine enzyme this parameter is markedly affected by the interaction with ligands which have a functional role, the constancy of the phosphorescence lifetime at various degrees of polymerization suggests that the association process is not accompanied by important conformational changes in the macromolecule.
Strambini
G B
GB
Istituto di Biofisica, Pisa, Italy.
Cioni
P
P
Felicioli
R A
RA
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008163:Luminescent Measurements; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan
D014364:Tryptophan; D000426:Alcohol Dehydrogenase; D005969:Glutamate Dehydrogenase