Abstract

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 pKa > 7, while the rate constant is pH-independent for substrates with a pKa < 7 suggesting that the pKas 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.