The Mechanism of O-Acetylserine Sulfhydrylase from Salmonella typhimurium

Summary

The pyridoxal 5′-phosphate dependent enzyme O-acetylserine sulfhydrylase catalyzes the final step in the biosynthesis of L-cysteine in enteric bacteria, from O-acetyl-L-serine (OAS) and inorganic sulfide. The enzyme has been studied in the absence and presence of reactants and reactant analogues using absorbance, fluorescence, circular dichroism, and ³¹P NMR spectroscopy. Addition of L-cysteine gives the external Schiff base, while addition of L-serine gives a mixture of the gem-diamine and the external Schiff base, and OAS gives the a-aminoacrylate intermediate. Formation of the external Schiff base with either cysteine or serine results in an increase in the energy transfer from an active site tryptophan to the protonated Schiff base as a result of an induced conformational change. Induced circular dichroism spectra of the bound cofactor indicate that a significant change in the orientation of the bound cofactor is realized when the a-aminoacrylate Schiff base is formed. The above data are corroborated by the ³¹P NMR spectra which suggest a further tightening of the binding of the cofactor in the cysteine external Schiff base, and an equilibrium between two species with serine present.