Time-resolved fluorescence of tryptophan synthase

Abstract

Time-resolved and steady-state fluorescence of the tryptophan synthase α 2 β 2 complex and of the β 2 dimer from Salmonella typhimurium were measured to characterize the conformational properties of the β subunit in the presence and in the absence of the α subunit when the catalytic species internal aldimine, external aldimine and α-aminoacrylate Schiff bases were selectively accumulated within the β active site. The fluorescence decay of the coenzyme pyridoxal 5′-phosphate, bound via a Schiff base in the β subunit of the α 2 β 2 complex (internal aldimine species), is accounted for by two lifetime (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 β 2 dimer are pH dependent, suggesting that the α-subunit binding alters the microenvironment of the β-subunit active site. This conclusion is also supported by the emission of the single tryptophanyl residue of the enzyme (Trp-177β). In the reaction of L-serine with the α 2 β 2 complex, the predominant catalytic intermediate is the external aldimine ( λ max = 422 nm) at pH 10, and the α-aminoacrylate ( λ 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 α 2 β 2 complex, at pH 10, and at a similar value in the β 2 dimer. The emission properties of the external aldimine with respect to the internal aldimine, and the small effects induced by α-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 α-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 α-aminoacrylate are present in single conformational states with distinct structural and/or dynamic properties that may modulate regulatory intersubunit signals.