Mechanism of the addition half of the O-acetylserine sulfhydrylase-A reaction

O-Acetylserine sulfhydrylase (OASS) catalyzes the last step in the cysteine biosynthetic pathway in enteric bacteria and plants, substitution of the β-acetoxy group of O-acetyl-L-serine (OAS) with inorganic bisulfide. The first half of the sulfhydrylase reaction, formation of the α-aminoacrylate intermediate, limits the overall reaction rate, while in the second half-reaction, with bisulfide as the substrate, chemistry is thought to be diffusion-limited. In order to characterize the second half-reaction, the pH dependence of the pseudo-first-order rate constant for disappearance of the α-aminoacrylate intermediate was measured over the pH range 6.0-9.5 using the natural substrate bisulfide, and a number of nucleophilic analogues. The rate is pH-dependent for substrates with a pK_a > 7, while the rate constant is pH-independent for substrates with a pK_a < 7 suggesting that the pK_aS of the substrate and an enzyme group are important in this half of the reaction. In D₂O, at low pD values, the amino acid external Schiff base is trapped, while in H₂O the reaction proceeds through release of the amino acid product, which is likely rate-limiting for all nucleophilic reactants. A number of new β-substituted amino acids were produced and characterized by ¹H NMR spectroscopy.