Peracchi A, Bettati S, Mozzarelli A, Rossi G L, Miles E W, Dunn M F
Istituto di Scienze Biochimiche, Università di Parma, Italy.
Biochemistry. 1996 Feb 13;35(6):1872-80. doi: 10.1021/bi951889c.
The equilibrium distribution of catalytic intermediates formed in the reaction of L-serine with the tryptophan synthase alpha 2 beta 2-complex from Salmonella typhimurium has been investigated by absorption and fluorescence spectroscopy as a function of pH, temperature, and alpha-subunit ligands. The novel result of this study is that the equilibrium between the two major catalytic species, the external aldimine and the alpha-aminoacrylate, is modulated by the ionization of two groups with apparent pK values of 7.8 +/- 0.3 and 10.3 +/- 0.2. Protonation of these groups stabilizes the alpha-aminoacrylate Schiff base by an estimated 100-fold with respect to the external aldimine. Furthermore, the formation of the alpha-aminoacrylate from the external aldimine is an endothermic process. Temperature slightly affects the apparent pK values but remarkably influences the amplitude of the phase associated with the ionization of each group. At 20 degrees C, each phase accounts for nearly half of the titration. Since the isolated beta 2-dimer does not exhibit a pH-dependent distribution of intermediates, the alpha-beta-subunit interactions seem critical to the onset of this functional property of the beta-subunit. The modulation of intersubunit interactions by the alpha-subunit ligands DL-alpha-glycerol 3-phosphate and phosphate leads to significant changes in the pH-dependent distribution of intermediates. At saturating concentrations of either of these alpha-subunit ligands, the alpha-aminoacrylate Schiff base is the predominant species over a wide pH range while the apparent pK values of the groups that control the equilibrium are not significantly affected. The pH-dependent interconversion of catalytic intermediates here reported has not been previously detected because phosphate buffers have usually been employed in the studies of this enzyme. Our findings are discussed in the light of a model in which specific protein conformations are associated with the external aldimine and the alpha-aminoacrylate Schiff bases, the latter being stabilized by temperature, protons, and alpha-subunit ligands.
利用吸收光谱和荧光光谱，研究了鼠伤寒沙门氏菌色氨酸合成酶α2β2复合物与L-丝氨酸反应中形成的催化中间体的平衡分布，该分布是pH、温度和α亚基配体的函数。本研究的新结果是，两种主要催化物种（外部醛亚胺和α-氨基丙烯酸酯）之间的平衡受两个基团电离的调节，这两个基团的表观pK值分别为7.8±0.3和10.3±0.2。相对于外部醛亚胺，这些基团的质子化使α-氨基丙烯酸酯席夫碱稳定了约100倍。此外，从外部醛亚胺形成α-氨基丙烯酸酯是一个吸热过程。温度对表观pK值影响较小，但对与每个基团电离相关的相的幅度影响显著。在20℃时，每个相几乎占滴定的一半。由于分离出的β2二聚体未表现出中间体的pH依赖性分布，α-β亚基相互作用似乎对β亚基这种功能特性的出现至关重要。α亚基配体DL-α-甘油3-磷酸和磷酸盐对亚基间相互作用的调节导致中间体pH依赖性分布发生显著变化。在这些α亚基配体中任一种的饱和浓度下，α-氨基丙烯酸酯席夫碱在很宽的pH范围内是主要物种，而控制平衡的基团的表观pK值没有受到显著影响。此处报道的催化中间体的pH依赖性相互转化以前未被检测到，因为在该酶的研究中通常使用磷酸盐缓冲液。我们根据一个模型讨论了这些发现，在该模型中，特定的蛋白质构象与外部醛亚胺和α-氨基丙烯酸酯席夫碱相关，后者通过温度、质子和α亚基配体得以稳定。
