Cook P F, Wedding R T
J Biol Chem. 1976 Apr 10;251(7):2023-9.
It has been determined from steady state kinetic studies using the sulfide ion selective electrode that O-acetylserine sulfhydrylase catalyzes a Bi Bi Ping Pong reaction between O-acetyl-L-serine and sulfide. Both O-acetyl-L-serine (OAS) and sulfide exhibit strong competitive substrate inhibition. A fit of all the data to the equation for the mechanism yields KOAS = 0.149 +/- 0.059 mM and KIOAS = 46.91 +/- 10.06 mM for O-acetyl-L-serine and KS2- = 0.066 +/- 0.004 mM and KIS2- = 0.013 +/- 0.006 mM for sulfide. Product inhibition studies varying either substrate at changing fixed levels of cysteine demonstrate that cysteine combines with enzyme at two places along the reaction sequence to produce inhibition with KiCys = 1.048 +/- 0.048 mM and KICys = 11.4 +/- 0.5 mM. Relatively high concentrations of acetate are required to produce inhibition and at least part of the acetate inhibition is due to ionic strength. However, the ability of acetate to reverse the spectral shift produced from the binding of O-acetyl-L-serine to enzyme and the isotope exchange between [14C]acetate and O-acetyl-L-serine does demonstrate that the O-acetyl-L-serine to acetate half-reaction is reversible. There is some doubt as to the specificity of acetate as a product inhibitor, since propionate can also be used to reverse the spectral shift. Spectral studies using ths spectral shift produced from binding O-acetyl-L-serine to enzyme confirms the assignment of a ping-pong mechanism since the spectral intermediate produced is alpha-aminoacrylic acid in Schiff base with pyridoxal phosphate and, therefore, the acetyl moiety has been beta eliminated. Isotope exchange has been demonstrated for both the O-acetyl-L-serine to acetate and sulfide to cysteine half-reactions which also confirms a ping-pong mechanism.
使用硫化物离子选择性电极进行的稳态动力学研究已确定，O-乙酰丝氨酸巯基酶催化O-乙酰-L-丝氨酸与硫化物之间的双底物双产物乒乓反应。O-乙酰-L-丝氨酸（OAS）和硫化物均表现出强烈的竞争性底物抑制作用。将所有数据拟合至该机制的方程，得出O-乙酰-L-丝氨酸的KOAS = 0.149±0.059 mM和KIOAS = 46.91±10.06 mM，硫化物的KS2- = 0.066±0.004 mM和KIS2- = 0.013±0.006 mM。在固定半胱氨酸水平变化时改变任一底物的产物抑制研究表明，半胱氨酸在反应序列中的两个位置与酶结合，产生抑制作用，其KiCys = 1.048±0.048 mM和KICys = 11.4±0.5 mM。需要相对高浓度的乙酸盐才能产生抑制作用，并且至少部分乙酸盐抑制作用是由于离子强度引起的。然而，乙酸盐能够逆转由O-乙酰-L-丝氨酸与酶结合产生的光谱位移以及[14C]乙酸盐与O-乙酰-L-丝氨酸之间的同位素交换，这确实证明了O-乙酰-L-丝氨酸到乙酸盐的半反应是可逆的。关于乙酸盐作为产物抑制剂的特异性存在一些疑问，因为丙酸盐也可用于逆转光谱位移。使用O-乙酰-L-丝氨酸与酶结合产生的光谱位移进行的光谱研究证实了乒乓机制的归属，因为产生的光谱中间体是与磷酸吡哆醛形成席夫碱的α-氨基丙烯酸，因此，乙酰基部分已被β消除。已证明O-乙酰-L-丝氨酸到乙酸盐和硫化物到半胱氨酸的半反应均存在同位素交换，这也证实了乒乓机制。
