Aspartic acid 214 in Citrobacter freundii tyrosine phenol-lyase ensures sufficient C--H-acidity of the external aldimine intermediate and proper orientation of the cofactor at the active site.
Aspartic acid 214 in Citrobacter freundii tyrosine phenol-lyase ensures sufficient C--H-acidity of the external aldimine intermediate and proper orientation of the cofactor at the active site.
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弗氏柠檬酸杆菌酪氨酸苯酚裂解酶中的天冬氨酸 214 确保外部醛亚胺中间体有足够的 C--H-酸性，并确保辅助因子在活性位点的正确方向。
DOI：
10.1016/j.bbapap.2006.05.001
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Phillips,RS
中科院分区：
文献类型：
--
作者：
Demidkina,TV;Faleev,NG;Papisova,AI;Bazhulina,NP;Kulikova,VV;Gollnick,PD;Phillips,RS
在酪氨酸酚裂解酶（TPL）的X射线结构中，Asp 214位于与辅因子的N1原子的氢键距离处。这个残基已被替换为丙氨酸和天冬酰胺和突变酶的特性进行了研究。取代导致辅因子亲和力降低约四个数量级。D214 A和D214 N TPL不催化1-Tyr和3-氟-1-Tyr的分解。它们分解底物，含有更好的离去基团，速率降低一到两个数量级。对数正态分辨率的突变酶的光谱显示，N1原子的辅因子是去质子化。内部和外部的醛亚胺的突变TPLs的光谱特性和它们与准底物的相互作用的数据表明，Asp 214的替代导致活性位点构象的改变。突变体酶与抑制剂作用后不形成显著量的醌，但在2 H2O中催化许多氨基酸的C-α-质子与氘的同位素交换。l-苯丙氨酸和3-氟-l-酪氨酸的同位素交换的kex值接近反应底物的kcat值。因此，对于突变型TPL，C-α-质子提取阶段可以被认为是整个反应的限速阶段。
In the X-ray structure of tyrosine phenol-lyase (TPL) Asp214 is located at H-bonding distance from the N1 atom of the cofactor. This residue has been replaced with Ala and Asn and the properties of the mutant enzymes have been studied. The substitutions result in a decrease in the cofactor affinity of about four orders of magnitude. D214A and D214N TPLs do not catalyze the decomposition of l-Tyr and 3-fluoro-l-Tyr. They decompose substrates, containing better leaving groups with rates reduced by one or two orders of magnitude. Lognormal resolution of the spectra of the mutant enzymes revealed that the N1 atom of the cofactor is deprotonated. Spectral characteristics of internal and external aldimines of the mutant TPLs and the data on their interaction with quasisubstrates demonstrate that replacements of Asp214 lead to alteration of active site conformations. The mutant enzymes do not form noticeable amounts of a quinonoid upon interaction with inhibitors, but catalyze isotope exchange of C–α-proton of a number of amino acids for deuterium in2H2O. The kexvalues for the isotope exchange of l-phenylalanine and 3-fluoro-l-tyrosine are close to the kcatvalues for reacting substrates. Thus, for the mutant TPLs the stage of C–α-proton abstraction may be considered as a rate-limiting for the whole reaction.