Phillips Robert S, Miles Edith W, McPhie Peter, Marchal Stephane, Georges Cédric, Dupont Yves, Lange Reinhard
Department of Chemistry, University of Georgia, Athens, Georgia 30602, USA.
J Am Chem Soc. 2008 Oct 15;130(41):13580-8. doi: 10.1021/ja8018466. Epub 2008 Sep 17.
Tryptophan synthase is an alpha2beta2 multienzyme complex that exhibits coupling of the alpha- and beta-subunit reactions by tightly controlled allosteric interactions. A wide range of parameters can affect the allosteric interactions, including monovalent cations, pH, alpha-site and beta-site ligands, temperature, and pressure. Rapid changes in hydrostatic pressure (P-jump) and temperature (T-jump) were used to examine the effects of pressure and temperature on the rates of the interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex. The intense fluorescence emission of the Tryptophan synthase L-Ser external aldimine complex at 495 nm, with 420 nm excitation, provides a probe of the conformational state of Trp synthase. P-jump measurements allowed the determination of rate constants for the reactions in the presence of Na(+), Na(+) with benzimidazole (BZI), and NH4(+). The data require a compressibility term, beta(o)(double dagger), to obtain good fits, especially for the NH4(+) and BZI/Na(+) data. The compressibility changes are consistent with changes in solvation in the transition state. The transition state for the relaxation is more similar in volume to the closed aminoacrylate complex in the presence of Na(+), while it is more similar to the open external aldimine in the presence of NH4(+). Differences between the relaxations for positive and negative P-jumps may arise from changing relative populations of microstates with pressure. T-jump experiments of the Na(+) form of the tryptophan synthase-L-Ser complex show large changes in rate and amplitude over the temperature range from 7 to 45 degrees C. The Arrhenius plots show strong curvature, and hence require a heat capacity term, DeltaC(p)(double dagger), to obtain good fits. The values of DeltaC(p)(double dagger) are very large and negative (-3.6 to -4.4 kJ mol(-1) K(-1)). These changes are also consistent with large changes in solvation in the transition state for interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex.
色氨酸合酶是一种α2β2多酶复合物，通过严格控制的变构相互作用实现α亚基和β亚基反应的偶联。多种参数可影响变构相互作用，包括单价阳离子、pH值、α位点和β位点配体、温度及压力。利用静水压力（P跳变）和温度（T跳变）的快速变化来研究压力和温度对色氨酸合酶-L-丝氨酸复合物中外醛亚胺和氨基丙烯酸酯中间体相互转化速率的影响。在420nm激发下，色氨酸合酶L-丝氨酸外醛亚胺复合物在495nm处有强烈的荧光发射，可作为色氨酸合酶构象状态的探针。P跳变测量确定了在存在Na⁺、Na⁺与苯并咪唑（BZI）以及NH₄⁺时反应的速率常数。数据需要一个压缩性项βₒ（双 dagger）才能得到良好拟合，特别是对于NH₄⁺和BZI/Na⁺数据。压缩性变化与过渡态溶剂化的变化一致。在存在Na⁺时，弛豫的过渡态在体积上更类似于封闭的氨基丙烯酸酯复合物，而在存在NH₄⁺时，它更类似于开放的外醛亚胺。正、负P跳变弛豫之间的差异可能源于随着压力微观状态相对数量的变化。色氨酸合酶-L-丝氨酸复合物Na⁺形式的T跳变实验表明，在7至45摄氏度的温度范围内，速率和幅度有很大变化。阿伦尼乌斯图显示出强烈的曲率，因此需要一个热容项ΔCₚ（双 dagger）才能得到良好拟合。ΔCₚ（双 dagger）的值非常大且为负（-3.6至-4.4kJ·mol⁻¹·K⁻¹）。这些变化也与色氨酸合酶-L-丝氨酸复合物中外醛亚胺和氨基丙烯酸酯中间体相互转化的过渡态溶剂化的巨大变化一致
