Structures of apo- and holo-tyrosine phenol-lyase reveal a catalytically critical closed conformation and suggest a mechanism for activation by K+ ions.
Structures of apo- and holo-tyrosine phenol-lyase reveal a catalytically critical closed conformation and suggest a mechanism for activation by K+ ions.
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DOI：
10.1021/bi0601858
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发表时间：
2006-06-20
期刊：
影响因子：
2.9
通讯作者：
Antson AA
中科院分区：
文献类型：
--
作者：
Milić D;Matković-Calogović D;Demidkina TV;Kulikova VV;Sinitzina NI;Antson AA
酪氨酸苯酚裂解酶是一种四聚体吡哆醛-5 ' -磷酸依赖酶，催化l-酪氨酸可逆水解裂解为苯酚和丙酮酸铵。在这里，我们以1.9 Å的分辨率描述了佛氏柠檬酸杆菌全酶的晶体结构。该结构揭示了蛋白质与辅因子吡哆醛-5 ' -磷酸相互作用的网络，以及催化重要的K+离子配位的细节。我们还以1.85 Å的分辨率给出了脱酶的结构。这两种结构都是用pH 8.0生长的晶体来确定的，pH接近最大酶活性的pH（8.2）。与先前在pH 6.0下测定的脱酶结构比较，发现有显著差异。结果表明，在pH 6.0时，酶活性降低可能是由于活性位点残基Tyr71、Tyr291、Arg381和单价阳离子结合残基Glu69的构象改变所致。此外，在pH 8.0时，我们观察到两种不同的活性位点构象：开放构象（之前表征过）和封闭构象（首次在β消除酶中观察到）。在封闭构象中，小结构域的很大一部分向大结构域进行了高达12 Å的异常运动，关闭了活性位点的间隙，并将催化重要的Arg381和Phe448带入活性位点。封闭的构象使以前的突变研究结果合理化，并表明观察到的活性位点关闭对酶反应过程和酶对其生理底物的特异性至关重要。最后，封闭的构象允许我们模拟酮（亚胺）类醌，关键的过渡中间体。
Tyrosine phenol-lyase, a tetrameric pyridoxal-5′-phosphate dependent enzyme, catalyses the reversible hydrolytic cleavage of l-tyrosine to phenol and ammonium pyruvate. Here we describe the crystal structure of the Citrobacter freundii holoenzyme at 1.9 Å resolution. The structure reveals a network of protein interactions with the cofactor, pyridoxal-5′-phosphate, and details of coordination of the catalytically important K+ ion. We also present the structure of the apoenzyme at 1.85 Å resolution. Both structures were determined using crystals grown at pH 8.0, which is close to the pH of the maximal enzymatic activity (8.2). Comparison of the apoenzyme structure with the one previously determined at pH 6.0 reveals significant differences. The data suggest that the decrease of the enzymatic activity at pH 6.0 may be caused by conformational changes in the active site residues Tyr71, Tyr291, Arg381 and in the monovalent cation binding residue Glu69. Moreover, at pH 8.0 we observe two different active-site conformations: open – which was characterised before, and closed – which is observed for the first time in β-eliminating lyases. In the closed conformation a significant part of the small domain undergoes an extraordinary motion of up to 12 Å towards the large domain, closing the active site cleft and bringing the catalytically important Arg381 and Phe448 into the active site. The closed conformation allows rationalisation of the results of previous mutational studies and suggests that the observed active-site closure is critical for the course of the enzymatic reaction and for the enzyme's specificity towards its physiological substrate. Finally, the closed conformation allows us to model keto(imino)quinonoid, the key transition intermediate.
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DOI：
10.1073/pnas.0335853100
发表时间：
2003-02-04
影响因子：
11.1
作者：
Brüggemann, H;Bäumer, S;Gottschalk, G
通讯作者：
Gottschalk, G
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
DOI：
10.1073/pnas.77.5.2559
发表时间：
1980-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
FORD, GC;EICHELE, G;JANSONIUS, JN
通讯作者：
JANSONIUS, JN
DOI：
10.1046/j.1432-1327.2000.01185.x
发表时间：
2000-03-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Bazhulina, NP;Morozov, YV;Demidkina, TV
通讯作者：
Demidkina, TV
DOI：
10.1080/00021369.1972.10860505
发表时间：
1972-01-01
期刊：
AGRICULTURAL AND BIOLOGICAL CHEMISTRY
影响因子：
--
作者：
ENEI, H;OKUMURA, S;MATSUI, H
通讯作者：
MATSUI, H