Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray structures of quinonoid intermediates.
Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray structures of quinonoid intermediates.
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DOI：
10.1074/jbc.m802061200
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发表时间：
2008-10-24
期刊：
影响因子：
--
通讯作者：
Antson AA
中科院分区：
文献类型：
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作者：
Milić D;Demidkina TV;Faleev NG;Matković-Calogović D;Antson AA
由许多PLP依赖性酶催化的氨基酸转化涉及从底物和辅因子之间形成的外部醛亚胺中提取Cα质子，从而形成喹啉中间体。尽管喹啉类中间体在PLP依赖性酶的催化作用中发挥了关键作用，但关于它们的结构的准确信息有限。我们用L-丙氨酸和L-甲硫氨酸捕获了弗氏柠檬酸杆菌酪氨酸酚裂解酶的喹喔啉中间体，并通过冷冻色谱法分别在1.9 μ m和1.95 μ m的分辨率下确定了它们的结构。这些数据揭示了蛋白质-PLP-底物相互作用的网络，其稳定了喹喔啉中间体的平面几何形状。在这两种结构中，蛋白质亚基有两种构象-开放和闭合，揭示了底物的结合和活性位点在其闭合过程中的重组保护喹喏酮中间体不受溶剂的影响，并将催化重要的残基带到适合在L-酪氨酸的β-消除过程中提取苯酚的位置的机制。此外，结构数据表明丙氨酸外消旋涉及两个碱基，赖氨酸257和水分子的机制。这两个碱基通过氢键系统连接，允许Cα质子的内部转移。
Amino acid transformations catalyzed by a number of PLP-dependent enzymes involve abstraction of the Cα proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. In spite of the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii tyrosine phenol-lyase with L-alanine and L-methionine in the crystalline state and determined their structures at 1.9 Å and 1.95 Å resolution, respectively, by cryocrystallography. The data reveal a network of protein–PLP–substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations – open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of phenol during the β-elimination of L-tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Cα proton.