TIME-RESOLVED FLUORESCENCE OF TRYPTOPHAN SYNTHASE

Time-resolved and steady-state fluorescence of the tryptophan synthase alpha(2) beta(2) complex and of the beta(2) dimer from Salmonella typ himurium were measured to characterize the conformational properties o f the beta subunit in the presence and in the absence of the alpha sub unit when the catalytic species internal aldimine, external aldimine a nd alpha-aminoacrylate Schiff bases were selectively accumulated withi n the beta active site. The fluorescence decay of the coenzyme pyridox al 5'-phosphate, bound via a Schiff base in the beta subunit of the al pha(2) beta(2) complex (internal aldimine species), is accounted for b y 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 proper ties of the internal aldimine in the beta(2) dimer are pH dependent, s uggesting that the alpha-subunit binding alters the microenvironment o f the beta-subunit active site. This conclusion is also supported by t he 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 (lambd a(max) = 422 nm) at pH 10, and the alpha-aminoacrylate (lambda(max) = 350 nm) at pH 7. The external aldimine exhibits a high fluorescence in tensity 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 respe ct to the internal aldimine, and the small effects induced by alpha-su bunit binding indicate a shielding of the coenzyme and a stabilization of its excited state. In contrast, the shea fluorescence lifetime (0. 4 ns) and the weak fluorescence emission of the alpha-aminoacrylate Sc hiff 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 w ith distinct structural and/or dynamic properties that may modulate re gulatory intersubunit signals.