Formation of the alpha-aminoacrylate immediate limits the overall reaction catalyzed by O-acetylserine sulfhydrylase.

O-Acetylserine sulfhydrylase-A (OASS-A) catalyzes the final step in the synthesis of L-cysteine, viz., the beta-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 alpha-aminoacrylate intermediate and acetate, is rate-limiting for the overall reaction catalyzed by OASS-A. No intermeidates are detected within the second half-reaction, and thus rate constants for all steps must be > or = 1000s-1 at the lowest sulfide concentration used. Within the first half reaction, formation of the extrernal Schiff base (Kassociation = 0.2 mM-1) is observed in the first milliseconds, followed by its slower conversion to the alpha-aminocacrylate 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-cysterine ot OASS-A results in a rapid formation of the external Schiff base (Kassociation = 6.7 mM-1), followed by transient formation of the alpha-aminoacylate intermediate with a slightly lower rate (70-100 s-1) compared to OAS. The alpha-aminoacrylate intermediate decays to generate a species absorbing maximally at 418 nm, resulting from attack of the cysteine thiol to give ether in external Schiff base linkage with the active site PLP.
AuthorsE U Woehl, C H Tai, M F Dunn, P F Cook
JournalBiochemistry (Biochemistry) Vol. 35 Issue 15 Pg. 4776-83 (Apr 16 1996) ISSN: 0006-2960 [Print] UNITED STATES
PMID8664267 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)
Chemical References
  • Schiff Bases
  • dehydroalanine
  • Cysteine Synthase
  • Alanine
  • Alanine (analogs & derivatives, biosynthesis)
  • Catalysis
  • Cysteine Synthase (metabolism)
  • Kinetics
  • Salmonella typhimurium (enzymology)
  • Schiff Bases
  • Spectrophotometry, Ultraviolet

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