Ground-State Destabilization by Phe-448 and Phe-449 Contributes to Tyrosine Phenol-Lyase Catalysis
Ground-State Destabilization by Phe-448 and Phe-449 Contributes to Tyrosine Phenol-Lyase Catalysis
复制标题
DOI：
10.1021/acscatal.6b01495
复制
发表时间：
2016-10-01
期刊：
影响因子：
12.9
通讯作者：
Hay, Sam
中科院分区：
文献类型：
--
作者：
Phillips, Robert S.;Vita, Andrew;Hay, Sam
鲍林提出的过渡态稳定在酶催化中的作用已经被广泛的研究清楚地证明了。相比之下，基态失稳也可以促进酶催化，但实验证据更加有限。近年来，人们获得了酶-底物复合体的高分辨X射线晶体结构，为基态应变提供了证据。我们发现Y71F和F448H突变的酪氨酸苯酚裂解酶(TPL)与底物3-氟-L酪氨酸形成络合物，底物芳香环在平面外弯曲约20度，这是TPL催化基态失稳的证据。在这里，我们现在已经定量地评估了基态失稳在TPL催化中的作用。Phe-448和Phe-449与结合的底物侧链密切接触，通过将这些残基突变为丙氨酸和亮氨酸，研究了它们通过基态失稳所起的作用。F448A、F448L和F449A TPL对L酪氨酸的脱酚活性分别为10~4、10~3和10~4倍，但它们与交替底物S-(邻硝基苯基)-L-半胱氨酸和S-乙基-L-半胱氨酸的活性接近正常。F448A TPL由L-酪氨酸和S-乙基-L-半胱氨酸形成醌类中间体，其速率常数与野生型TPL相似。此外，F448A TPL能由S-乙基-L-半胱氨酸生成氨基丙烯酸酯中间体，而不能由L-酪氨酸生成，其反应速率常数与野生型TPL相似。因此，该突变对L酪氨酸中苯酚的清除具有特异性。考察了静水压力对F448H、F448A、TPL和3-氟-L酪氨酸合成苯醌中间体的速率和平衡的影响。尽管最快的相只显示了较小的压力影响，但三个较慢的相具有显著的压力依赖性，这表明它们可能与构象变化有关。这些结果表明，Phe-448和Phe-449通过在L酪氨酸底物中引入基态失稳，总共对第三方物流的催化作用贡献了约108个基态，约占估计速率加速的50%。
The role of transition-state stabilization in enzyme catalysis, as proposed by Pauling, has been clearly demonstrated by extensive studies. In contrast, ground-state destabilization can also contribute to enzyme catalysis, but experimental evidence has been more limited. In recent years, high-resolution X-ray crystal structures of enzyme-substrate complexes have been obtained which show evidence for ground-state strain. We found that Y71F and F448H mutant tyrosine phenol-lyase (TPL) form complexes with 3-fluoro-L-tyrosine, a substrate, which shows a bending of the substrate aromatic ring about 20 degrees out of plane, and we suggested that this was evidence for ground-state destabilization in TPL catalysis. Here, we have now evaluated quantitatively the role of ground-state destabilization in TPL catalysis. Phe-448 and Phe-449 are in close contact with the bound substrate side chain, and by mutating these residues to alanine and leucine, the contribution they play via ground-state destabilization was investigated. F448A, F448L and F449A TPL have activity for elimination of phenol from L-tyrosine reduced by a factor of 104, 103, and 104, respectively, but they have near-normal activity with the alternate substrates S-(o-nitrophenyl)-L- cysteine and S-ethyl-L-cysteine. F448A TPL forms quinonoid intermediates from L-tyrosine and S-ethyl-L-cysteine with rate constants similar to those of wild-type TPL. In addition, F448A TPL can form an aminoacrylate intermediate from S-ethyl-L-cysteine but not L-tyrosine, with a rate constant similar to that of wild-type TPL. Thus, the effect of the mutation is specifically on the elimination of phenol from L-tyrosine. We also examined the effect of hydrostatic pressure on the rates and equilibria of formation of the quinonoid intermediates from F448H and F448A TPL and 3-fluoro-L-tyrosine. Although the fastest phase shows only a small effect of pressure, the three slower phases have significant pressure dependences, suggesting that they may be associated with a conformational change. These results demonstrate that Phe-448 and Phe-449 contribute a total of about 108 to catalysis in TPL, about 50% of the estimated rate acceleration, by introducing ground-state destabilization into the L-tyrosine substrate.