Williams J C, McDermott A E
Department of Chemistry, Columbia University, New York, New York 10027, USA.
Biochemistry. 1995 Jul 4;34(26):8309-19. doi: 10.1021/bi00026a012.
Using solid-state deuterium NMR, we have measured the motion of the flexible loop of triosephosphate isomerase (TIM) with and without substrate and transition-state analogs. The measurements were carried out on a catalytically competent mutant of TIM W90Y W157F containing a single tryptophan (W168) in the flexible loop; W168 is the only strictly conserved tryptophan in the currently available TIM sequences. The solid-state NMR samples were prepared by precipitation using polyethylene glycol, and kinetic analysis of the PEG-precipitated TIM gave values for kcat, Km, and KI similar to those measured in solution for the substrate and substrate and transition-state analogs. Deuterium NMR spectra of samples prepared with tryptophan labeled at the indole positions with and without any substrate or analogs indicate that the loop jumps between two conformations at a rate of 3 x 10(4) s-1 (from the predominant to the less populated form) with a population ratio of 10:1. Surprisingly, spectra of TIM ligated with a substrate analog, glycerol 3-phosphate (G3P), or with a tight-binding transition-state analog, phosphoglycolate (PGA), show that the loop moves with a rate similar to the rate in the empty enzyme and also has a similar population ratio for the two conformers. This observation indicates that loop closure is not ligand gated but is a natural motion of the protein. Furthermore, the measured rate is approximately matched to the turnover time. We did not observe a signal for TIM labeled with alpha-deuteriotryptophan, although it was prepared in a fashion analogous to the ring-labeled sample and had a specific activity and protein concentration comparable to the latter. For this deuterium concentration, we would expect to observe the NMR signal unless the deuterium relaxation were very slow. The hypothesis that the spin-lattice relaxation of the alpha-deuteron is very slow would be consistent with the observed dynamics of the ring-deuterated TIM.
我们使用固态氘核磁共振技术，测量了磷酸丙糖异构酶（TIM）柔性环在有和没有底物及过渡态类似物情况下的运动。测量是在TIM的催化活性突变体W90Y W157F上进行的，该突变体在柔性环中含有一个色氨酸（W168）；W168是目前可用的TIM序列中唯一严格保守的色氨酸。固态核磁共振样品通过聚乙二醇沉淀制备，对聚乙二醇沉淀的TIM进行动力学分析得到的kcat、Km和KI值，与在溶液中对底物以及底物和过渡态类似物测量得到的值相似。用吲哚位置标记色氨酸且有和没有任何底物或类似物的样品的氘核磁共振谱表明，该环以3×10⁴ s⁻¹的速率在两种构象之间跳跃（从主要构象到较少占据的构象），占据比例为10:1。令人惊讶的是，与底物类似物甘油3 - 磷酸（G3P）或紧密结合的过渡态类似物磷酸乙醇酸（PGA）结合的TIM的谱表明，该环的运动速率与无配体酶中的速率相似，并且两种构象的占据比例也相似。这一观察结果表明，环的闭合不是由配体控制的，而是蛋白质的一种自然运动。此外，测得的速率与周转时间大致匹配。我们没有观察到用α - 氘代色氨酸标记的TIM的信号，尽管它是以与环标记样品类似的方式制备的，并且比活性和蛋白质浓度与后者相当。对于这种氘浓度，除非氘弛豫非常慢，我们预计会观察到核磁共振信号。α - 氘核的自旋 - 晶格弛豫非常慢这一假设与观察到的环氘代TIM的动力学是一致的。
