Characterization of tryptophan and coenzyme luminescence in tryptophan synthase from Salmonella typhimurium.
Tryptophan synthase from Salmonella typhimurium is a bifunctional alpha 2 beta 2 complex that catalyzes the formation of L-tryptophan. We have characterized over the temperature range from 160 to 293 K the fluorescence and phosphorescence properties of the single tryptophan present at position 177 of the beta-subunit and of the pyridoxal 5'-phosphate bound through a Schiff's base in the beta-active site. The comparison between the fluorescence of the pyridoxal phosphate bound either to the protein or to valine free in solution indicates substantial protection for the coenzyme against thermal quenching and a greater intensity of the ketoenamine tautomer band. Trp-177 is highly luminescent, and its proximity to the pyridoxal moiety leads to an over 50% quenching of its fluorescence with both reduced and native coenzyme. The Trp phosphorescence spectrum possesses a narrow, well-defined, 0-0 vibrational band centered at 418.5 nm, a wavelength that indicates strong polar interactions with neighboring charges. The observation of delayed fluorescence in the native complex implies that the excited triplet state is involved in a process of triplet-singlet energy transfer to the ketoenamine tautomer. The rate of energy transfer, heterogeneous in low-temperature glasses with rate constants of 2.26 and 0.07 s-1, becomes homogeneous in fluid solutions as the coenzyme tautomer interconversion is likely faster than the phosphorescence decay. In both apo- and holo-alpha 2 beta 2, the phosphorescence from Trp-177 is long-lived even at ambient temperature.(ABSTRACT TRUNCATED AT 250 WORDS)
Strambini
G B
GB
CNR-Istituto di Biofisica, Pisa, Italy.
Cioni
P
P
Peracchi
A
A
Mozzarelli
A
A
D001665:Binding Sites; D008163:Luminescent Measurements; D046911:Macromolecular Substances; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000032:analysis / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology*; D012545:Schiff Bases; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan / Q000378:metabolism*; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000378:metabolism*
D046911:Macromolecular Substances; D012545:Schiff Bases; D011732:Pyridoxal Phosphate; D014364:Tryptophan; D014367:Tryptophan Synthase