Woehl E U, Tai C H, Dunn M F, Cook P F
Department of Biochemistry, University of California, Riverside, 92521-0129, USA.
Biochemistry. 1996 Apr 16;35(15):4776-83. doi: 10.1021/bi952938o.
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.
O-乙酰丝氨酸巯基酶A（OASS-A）催化L-半胱氨酸合成的最后一步，即通过乒乓动力学机制，硫化物对O-乙酰-L-丝氨酸（OAS）中的乙酸盐进行β取代。进行了快速扫描停流、单波长吸光度和荧光停流实验，以获取有关OASS-A整体反应和各个半反应的限速步骤的位置和限制量的信息。第一个半反应，即OAS转化为α-氨基丙烯酸酯中间体和乙酸盐，是OASS-A催化的整体反应的限速步骤。在第二个半反应中未检测到中间体，因此在使用的最低硫化物浓度下，所有步骤的速率常数必须≥1000 s⁻¹。在第一个半反应中，在最初的几毫秒内观察到外部席夫碱的形成（缔合常数Kassociation = 0.2 mM⁻¹），随后其以300 s⁻¹的速率常数较慢地转化为α-氨基丙烯酸酯中间体，接近V/Et所获得的130 s⁻¹的值[Tai, C.H., Nalabolu, S.R., Jacobson, T.M., Minter D.E., & Cook, P.F. (1993) Biochemistry 32, 6433 - 6442]。向OASS-A中添加L-半胱氨酸会导致外部席夫碱迅速形成（缔合常数Kassociation = 6.7 mM⁻¹），随后与OAS相比，α-氨基丙烯酸酯中间体的瞬时形成速率略低（70 - 100 s⁻¹）。α-氨基丙烯酸酯中间体衰变生成在418 nm处具有最大吸收的物质，这是由于半胱氨酸硫醇的攻击导致与活性位点PLP形成外部席夫碱键的醚。
