Conformational probes of O-acetylserine sulfhydrylase: fluorescence of tryptophans 50 and 161

O-acetylserine sulfhydrylase-A is a dimeric pyridoxal 5'-phosphate-dependen t enzyme catalyzing the synthesis of L-cysteine. We have characterized the fluorescence properties of the two tryptophans of the enzyme, residues Trp 50 and 161, in the native state and after the binding of substrates and pro ducts, as probes of the conformational changes that take place in the apo- to holo-enzyme transformation and during the catalytic process. Upon excita tion at 298 nm, the emissions of the apo- and holo-enzyme are centered at 3 43 and 338 nm, respectively, and are characterized by biexponential decays. The emission of the holo-enzyme is reduced by about 60% with respect to th at of the apo-enzyme. The deconvolution of the peaks and the time-resolved fluorescence data indicate that (i) the emission of Trp 50 is centered at 3 42 and 333 nm in the apo- and holo-enzyme, respectively; (ii) the emission of Trp 161 is centered at 357 nm in both the apo- and holo-enzyme; (iii) an energy transfer process quenches the fluorescence of the two tryptophan re sidues, being more efficient for Trp 50, which is less exposed to the solve nt; (iv) the quenching of Trp 161 emission is mainly due to conformational changes accompanying the apo- to holo-enzyme transition. Steady-state and t ime-resolved fluorescence, collected in the presence of the products acetat e or L-cysteine, the product analogue L-serine, and the substrate O-acetyls erine or its analogue beta-chloro-alanine, indicate that tryptophan emissio ns are significantly affected by variations of the equilibrium distribution between the enolimine and ketoenamine tautomers, which are differently pop ulated during catalysis. (C) 1999 Elsevier Science S.A. All rights reserved .