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

Abstract O -acetylserine sulfhydrylase-A is a dimeric pyridoxal 5′-phosphate-dependent 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 products, as probes of the conformational changes that take place in the apo- to holo-enzyme transformation and during the catalytic process. Upon excitation at 298 nm, the emissions of the apo- and holo-enzyme are centered at 343 and 338 nm, respectively, and are characterized by biexponential decays. The emission of the holo-enzyme is reduced by about 60% with respect to that of the apo-enzyme. The decon volution of the peaks and the time-resolved fluorescence data indicate that (i) the emission of Trp 50 is centered at 342 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 residues, being more efficient for Trp 50, which is less exposed to the solvent; (iv) the quenching of Trp 161 emission is mainly due to conformational changes accompanying the apo- to holo-enzyme transition. Steady-state and time-resolved fluorescence, collected in the presence of the products acetate or L -cysteine, the product analogue L -serine, and the substrate O -acetylserine or its analogue β-chloro-alanine, indicate that tryptophan emissions are significantly affected by variations of the equilibrium distribution between the enolimine and ketoenamine tautomers, which are differently populated during catalysis.