title: Time-resolved fluorescence of tryptophan synthase.
abstract: Time-resolved and steady-state fluorescence of the tryptophan synthase alpha 2 beta 2 complex and of the beta 2 dimer from Salmonella typhimurium were measured to characterize the conformational properties of the beta subunit in the presence and in the absence of the alpha subunit when the catalytic species internal aldimine, external aldimine and alpha-aminoacrylate Schiff bases were selectively accumulated within the beta active site. The fluorescence decay of the coenzyme pyridoxal 5'-phosphate, bound via a Schiff base in the beta subunit of the alpha 2 beta 2 complex (internal aldimine species), is accounted for by two lifetimes (2.9 and 0.9 ns) of almost equal fractional intensity that are slightly affected by pH. Accordingly, both the absorption and emission spectra were found to be pH independent. The emission properties of the internal aldimine in the beta 2 dimer are pH dependent, suggesting that the alpha-subunit binding alters the microenvironment of the beta-subunit active site. This conclusion is also supported by the emission of the single tryptophanyl residue of the enzyme (Trp-177 beta). In the reaction of L-serine with the alpha 2 beta 2 complex, the predominant catalytic intermediate is the external aldimine (lambda(max) = 422 nm) at pH 10, and the alpha-aminoacrylate (lambda(max) = 350 nm) at pH 7. The external aldimine exhibits a high fluorescence intensity at 500 nm that decays with a single lifetime of 6.2 ns in the alpha 2 beta 2 complex, at pH 10, and at a similar value in the beta 2 dimer. The emission properties of the external aldimine with respect to the internal aldimine, and the small effects induced by alpha-subunit binding indicate a shielding of the coenzyme and a stabilization of its excited state. In contrast, the short fluorescence lifetime (0.4 ns) and the weak fluorescence emission of the alpha-aminoacrylate Schiff base indicate an increase of non-radiative processes possibly due to a more tight coupling of this intermediate with the protein matrix with respect to the external aldimine. Whereas the internal aldimine is distributed in two tautomeric forms, both the external aldimine and the alpha-aminoacrylate are present in single conformational states with distinct structural and/or dynamic properties that may modulate regulatory intersubunit signals.
authors.affiliation: Institute of Physical Sciences, University of Parma, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Vaccari
authors.forename: S
authors.initials: S
authors.lastname: Benci
authors.forename: A
authors.initials: A
authors.lastname: Peracchi
authors.forename: A
authors.initials: A
authors.lastname: Mozzarelli
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D046911:Macromolecular Substances; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology; D012694:Serine / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D014367:Tryptophan Synthase / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D046911:Macromolecular Substances; D012694:Serine; D014367:Tryptophan Synthase