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.
Woehl
E U
EU
Department of Biochemistry, University of California, Riverside, 92521-0129, USA.
Tai
C H
CH
Dunn
M F
MF
Cook
P F
PF
D000409:Alanine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D002384:Catalysis; D003547:Cysteine Synthase / Q000378:metabolism*; D007700:Kinetics; D012486:Salmonella typhimurium / Q000201:enzymology*; D012545:Schiff Bases; D013056:Spectrophotometry, Ultraviolet
D012545:Schiff Bases; C015102:dehydroalanine; D003547:Cysteine Synthase; D000409:Alanine