Formation of the α-Aminoacrylate Intermediate Limits the Overall Reaction Catalyzed by O-Acetylserine Sulfhydrylase

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Abstract
O-Acetylserine sulfhydrylase-A (OASS-A) catalyzes the final step in the synthesis of l-cysteine, viz., the β-substitution of acetate in O-acetyl-l-serine (OAS) by sulfide via a ping-pong kinetic mechanism. Rapid-scanning stopped-flow and single-wavelength absorbance and fluorescence stopped-flow experiments were carried out to obtain information on the location and amount of limitation of rate-determining steps for the overall reaction and the individual half-reactions of OASS-A. The first half-reaction, conversion of OAS to the α-aminoacrylate intermediate and acetate, is rate-limiting for the overall reaction catalyzed by OASS-A. No intermediates are detected within the second half-reaction, and thus rate constants for all steps must be ≥1000 s-1 at the lowest sulfide concentration used. Within the first half reaction, formation of the external Schiff base (Kassociation = 0.2 mM-1) is observed in the first few milliseconds, followed by its slower conversion to the α-aminoacrylate intermediate with a rate constant of 300 s-1, close to the value of 130 s-1 obtained for V/Et [Tai, C.-H., Nalabolu, S. R., Jacobson, T. M., Minter, D. E., & Cook, P. F. (1993) Biochemistry 32, 6433−6442]. Addition of l-cysteine to OASS-A results in a rapid formation of the external Schiff base (Kassociation = 6.7 mM-1), followed by transient formation of the α-aminoacrylate intermediate with a slightly lower rate (70−100 s-1) compared to OAS. The α-aminoacrylate intermediate decays to generate a species absorbing maximally at 418 nm, resulting from attack of the cysteine thiol to give a thiol ether in external Schiff base linkage with the active site PLP.