Hwang C C, Woehl E U, Minter D E, Dunn M F, Cook P F
Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center at Fort Worth 76107-2699, USA.
Biochemistry. 1996 May 21;35(20):6358-65. doi: 10.1021/bi9602472.
Primary and alpha-secondary deuterium kinetic isotope effects have been measured for the O-acetylserine sulfhydrylase from Salmonella typhimurium using both steady-state and single-wavelength stopped-flow studies. Data suggest an asymmetric transition state for alpha-proton abstraction by the active site lysine and the elimination of the acetyl group of O-acetyl-L-serine (OAS) to form the alpha-aminoacrylate intermediate. The value of D(V/KOAS) using OAS-2-d is dependent on pH from 5.8 to 7.0 with independent values of 2.8 and 1.7 estimated at low and high pH, respectively. Thus, OAS is sticky, and a value of 1.5 is calculated for the forward commitment to catalysis, indicating that the OAS external Schiff base preferentially partitions toward the alpha-aminoacrylate intermediate compared to OAS being released from enzyme. The intrinsic primary deuterium isotope effect determined from single-wavelength stopped-flow studies of alpha-proton abstraction by the active site lysine is about 2.0. D(V/KOAS) and T(V/KOAS) were determined as 2.6 +/- 0.1 and 4.2 +/- 0.2 at pH 6.1, respectively, giving a calculated intrinsic deuterium isotope effect of 3.3 +/- 0.9, consistent with the D(V/KOAS) obtained from steady-state studies at low pH. The alpha-secondary deuterium kinetic isotope effect using OAS-3,3-d2 is 1.11 +/- 0.06 obtained by direct comparison of initial velocities and 1.2 obtained by single-wavelength stopped-flow experiments. Data can be compared to a value of 1.81 +/- 0.04 using OAS-3,3-d2 for alpha-DKeq for the first half-reaction.
利用稳态和单波长停流研究，测定了鼠伤寒沙门氏菌中O-乙酰丝氨酸巯基酶的一级和α-二级氘动力学同位素效应。数据表明，活性位点赖氨酸夺取α-质子以及消除O-乙酰-L-丝氨酸（OAS）的乙酰基团以形成α-氨基丙烯酸酯中间体的过渡态是不对称的。使用OAS-2-d时，D(V/KOAS)的值在pH 5.8至7.0范围内依赖于pH，在低pH和高pH下分别估计为2.8和1.7。因此，OAS具有粘性，催化正向的贡献值计算为1.5，这表明与从酶中释放的OAS相比，OAS外部席夫碱优先向α-氨基丙烯酸酯中间体分配。通过单波长停流研究活性位点赖氨酸夺取α-质子确定的内在一级氘同位素效应约为2.0。在pH 6.1时，D(V/KOAS)和T(V/KOAS)分别测定为2.6±0.1和4.2±0.2，计算得到的内在氘同位素效应为3.3±0.9，与在低pH下稳态研究获得的D(V/KOAS)一致。使用OAS-3,3-d2的α-二级氘动力学同位素效应通过初始速度的直接比较得到的值为1.11±0.06，通过单波长停流实验得到的值为1.2。该数据可与使用OAS-3,3-d2的α-DKeq用于第一个半反应的值1.81±0.04进行比较。
