Allosteric regulation of tryptophan synthase: Effects of pH, temperature, and alpha-subunit ligands on the equilibrium distribution of pyridoxal 5'-phosphate-L-serine intermediates
Allosteric regulation of tryptophan synthase: Effects of pH, temperature, and alpha-subunit ligands on the equilibrium distribution of pyridoxal 5'-phosphate-L-serine intermediates
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DOI：
10.1021/bi951889c
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发表时间：
1996-02-13
期刊：
影响因子：
2.9
通讯作者：
Dunn, MF
中科院分区：
文献类型：
--
作者：
Peracchi, A;Bettati, S;Dunn, MF
通过吸收和荧光光谱法研究了L-丝氨酸与鼠伤寒沙门氏菌色氨酸合成酶α（2）β（2）复合物反应中形成的催化中间体的平衡分布，这些中间体是pH、温度和α-亚基配体的函数。本研究的新结果是，两个主要的催化物种之间的平衡，外部醛亚胺和α-氨基丙烯酸酯，调制的两个基团的电离与表观pK值为7.8 +/- 0.3和10.3 +/- 0.2。这些基团的质子化使α-氨基丙烯酸酯席夫碱相对于外部醛亚胺稳定估计100倍。此外，由外部醛亚胺形成α-氨基丙烯酸酯是吸热过程。温度轻微影响表观pK值，但显着影响与电离的每个基团的相位的振幅。在20 ℃时，每个相占滴定的近一半。由于分离的β（2）-二聚体不显示中间体的pH依赖性分布，因此α-β-亚基相互作用似乎对β-亚基的这种功能性质的发生至关重要。α-亚基配体DL-α-甘油3-磷酸和磷酸对亚基间相互作用的调节导致中间体的pH依赖性分布发生显著变化。在饱和浓度的这些α-亚基配体，α-氨基丙烯酸酯席夫碱是在一个很宽的pH范围内的主要物种，而控制平衡的基团的表观pK值没有显着影响。本文报道的催化中间体的pH依赖性相互转化以前没有被检测到，因为磷酸盐缓冲液通常被用于这种酶的研究。我们的研究结果进行了讨论，在一个模型中，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，后者被稳定的温度，质子，和α-亚基配体。
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 a-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.